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Inhibition of ethylene production by rhizobitoxine.

Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorghum was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, alpha-keto-gamma-methylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.Incorporation of (14)C from added methionine-(14)C into ethylene was curtailed by rhizobitoxine to about the same extent as was ethylene production. These results suggest that rhizobitoxine interferes with ethylene biosynthesis by blocking the conversion of methionine to ethylene and not indirectly by inhibiting the biosynthesis of methionine. Ethylene production by Penicillium digitatum, a fungus which produces ethylene via pathways not utilizing methionine as a precursor, was not affected by rhizobitoxine.

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Auxin-induced Conjugation Systems in Peas.

Pretreatment of pea (Pisum sativum var. Alaska) sections with any active auxin induces an enzyme which forms aspartate conjugates of exogenously supplied indoleacetic acid, naphthaleneacetic acid, or benzoic acid. Whereas induction of this system is an absolutely auxin-specific process, another enzyme, which forms benzoylmalic acid, is induced both by auxins and by physiologically inactive aromatic carboxylic acids. Induction of both enzymes is abolished by low levels of RNA and protein synthesis inhibitors. The induction specificities and other characteristics of the two systems are compared.

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Relation of Phytochrome-enhanced Geotropic Sensitivity to Ethylene Production.

Brief exposure of etiolated pea (Pisum sativum cv. Alaska) seedlings to red light enhances subsequent development of geotropic curvature of the stem. Both this response and inhibition of ethylene production by red light become maximal 8 hours after illumination. Very low concentrations of applied ethylene inhibit development of geotropic curvature, whereas hypobaric treatment enhances geotropic sensitivity by removing endogenous ethylene. Increased geotropic sensitivity after illumination is accompanied by increased lateral migration of (3)H-indoleacetic acid in response to gravity, and ethylene inhibits this lateral migration. It is suggested, therefore, that red light-enhanced geotropic sensitivity is caused by increased lateral auxin transport resulting from a reduction in ethylene production after illumination.

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The Regulatory Properties of Purified Phaseolus aureus Sucrose Synthetase.

Phaseolus aureus sucrose synthetase, purified to homogeneity, was assayed in the presence of a variety of biological compounds to test for possible regulatory effectors. The oxidized form of nicotinamide adenine dinucleotide phosphate, as well as indoleacetic acid, gibberellic acid, and pyrophosphate were found to activate the forward reaction (sucrose degradation) and inhibit the reverse reaction (sucrose synthesis). The reduced form of nicotinamide adenine dinucleotide phosphate antagonizes the effect of the oxidized form. Fructose 1-phosphate and divalent cations inhibit the forward and activate the reverse reaction. Pyrophosphate and fructose 1-phosphate are effective only in the presence of magnesium chloride. Uridine triphosphate inhibits both the forward and reverse reactions. All effectors except gibberellic acid are active only in the millimolar range of concentrations; maximal stimulation for any effector is approximately 2-fold. The effects of combinations of effectors are roughly additive. Using pyrophosphate in the presence of magnesium chloride as an effector, results of kinetic studies offer a model by which an effector can activate an enzymatic reaction in one direction and inhibit in the reverse direction.

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Some factors regulating auxin translocation in intact bean seedlings.

Several factors which influence the translocation patterns of stem-injected indoleacetic acid, 2,4-dichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxyacetic acid in bean seedlings (Phaseolus vulgaris L. cv. Stringless Greenpod) were characterized. The acropetal translocation of auxin from the site of injection is markedly sensitive to concentration in the range of 1.0 to 5.0 micrograms per plant. The antiauxin p-chlorophenoxyisobutyric acid enhanced translocation of 2,4,5-trichlorophenoxyacetic acid to the growing shoots and primary leaves. Translocation to the roots was unaffected by p-chlorophenoxyisobutyric acid while leaching of 2,4,5-trichlorophenoxyacetic acid into the nutrient solution was enhanced slightly. Steam girdling experiments revealed that translocation to the primary leaves was in the xylem. The protein synthesis inhibitor, cycloheximide, inhibited accumulation of 2,4,5-trichlorophenoxyacetic acid in young shoots, epicotyls, and roots and enhanced accumulation in the primary leaves. The relative exchangeability of auxin between xylem and phloem is discussed in terms of regulation of auxin movements in intact bean seedlings.

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Effects of calcium and kinetin on growth and cell wall composition of pea epicotyls.

Kinetin and CaCl(2), in the presence of indoleacetic acid, promoted lateral expansion of epicotyls of decapitated and derooted Alaska pea seedlings (Pisum sativum L.) and inhibited their elongation. This growth response was correlated with the development of cell walls unusually rich in pectic uronic acids. Epicotyls in calcium-auxin solutions continued to enlarge and to add new wall material long after tissues in auxin only had stopped. Longitudinal enlargement, associated with the development of walls poor in pectic uronic acids, was favored by KCl, MgCl(2), and ethylenediaminetetraacetate. The last of these agents promoted the loss of (45)Ca from the epicotyls. Seedings grown in vermiculite moistened with CaCl(2), KCl, or MgCl(2) solutions did not differ in appearance or in the composition of their walls. They responded similarly to experimental treatment except that the decapitated epicotyls of the MgCl(2)-grown plants suffered an absolute loss of pectic uronate when incubated in that salt.

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Kinetics of stress relaxation properties of oat coleoptile cell wall after geotropic stimulation.

This study describes the stress relaxation of the cell wall of oat (Avena sativa) coleoptiles after different periods of geotropic stimulation. The upper and lower tissues (with respect to gravity) of geotrophically stimulated coleoptiles exhibit different wall properties. The lower tissues are less resistant to deformation than the upper. The ratio of stress to strain is significatly less in the lower than in the upper tissue. Similarly, the relaxation time and the minimum relaxation time, derived from the Maxwell model which describes the physical characteristics of polymers, are also shorter in the lower tissue. However, the maximum relaxation time shows no difference between the upper and lower tissues of a geotropically stimulated coleoptile. The differences between the tissues begin at about 8 minutes after the commencement of stimulation, similar to the time for the initiation of dictyosome redistribution, and precede the onset of geotropism. The above responses of the cell wall of the lower tissue are similar to those induced by indoleacetic acid. The parameters of wall properties of the coleoptiles of both the control and the geostimulated fluctuate rhythmically with time. The periodic changes in wall properties of the coleoptile are compared to other cyclic physiological phenomena.

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Regulation of beta-Glucan Synthetase Activity by Auxin in Pea Stem Tissue: I. Kinetic Aspects.

Treatment of pea stem segments with indoleacetic acid (IAA) causes within 1 hour a 2- to 4-fold increase in activity of particulate uridine diphosphoglucose-dependent beta-glucan synthetase obtainable from the tissue. The IAA effect is observable in tissue from all parts of the elongation zone of the pea stem, and also in older tissue that is not capable of a cell enlargement response to IAA. A large increase in activity is caused by IAA only if synthetase activity in the isolated tissue has first been allowed to fall substantially below the intact plant level, and only if sucrose is supplied along with IAA. Treatment of tissue with sucrose alone after a period of sugar starvation causes a transient rise of synthetase activity. The decline in synthetase activity in absence of IAA, the rise caused by IAA, and the transient rise caused by sucrose are all strongly temperature-dependent. IAA and sucrose do not affect the activity of isolated synthetase particles. Synthetase activity in vivo is sensitive to as low as 0.1 mum IAA and is increased by IAA analogues that are active as auxins on elongation but not by nonauxin analogues. Activity begins to rise 10 to 15 minutes after exposure to IAA, which places this among the most rapid enzyme effects of a plant growth regulator heretofore demonstrated, and among the most rapid known metabolic effects of auxins. The effect is seen also with polysaccharide synthetase activity using uridine diphosphate-galactose or uridine diphosphate-xylose as substrates, and to a lesser extent with guanosine diphosphoglucose-dependent glucan synthetase activity. Glucan synthetase from IAA-treated tissue appears to have a higher affinity for uridine diphosphate-glucose than the control.

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A role for calcium in auxin transport.

The basipetal transport of the auxin, indoleacetic acid, in sunflower stem sections is markedly suppressed by washing the tissue in ethylenediaminetetraacetate, and transport is restored by subsequent application of calcium solutions. The ethylenediaminetetraacetate treatment is shown to result in the removal of substantial amounts of calcium from the tissue, and the restoration of transport is distinctive for calcium solutions, lesser effects being observed for magnesium and lanthanum, and little effect for monovalent cations. The calcium effects are interpreted as indicating that the auxin transport system depends upon structural or functional features of cellular membranes which involve calcium in a manner analogous to the transport of inorganic ions.

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Polarity and rate of transport of cyclic adenosine 3,5'-monophosphate in the coleoptile.

Transport of tritiated cyclic AMP in the coleoptile of oats (Avena sativa) and corn (Zea mays) is polar, with basipetal to acropetal ratios of 4.0 and 3.2, respectively. The rate of transport is approximately that of indoleacetic acid. The linear velocity of transport, however, is at least five times that of auxin. A loss in transport polarity of the nucleotide occurs in subapical tissues within several hours after decapitation of the coleoptile, accompanied by a decrease in transport rate. The loss in polarity is not reversed by exogenous auxin, but the reduction in transport is. Auxin also inhibits the uptake of cyclic AMP. Exogenous cyclic AMP is metabolized rapidly by coleoptile tissues. If cyclic AMP does have a cellular function in the coleoptile, its transport behavior is compatible with that of a hormone.

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Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L.

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10(-5) or 10(-2)m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10(-2)m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10(-5)m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.

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Localization of Stem Elongation Control in Cucumis sativus L.

Reciprocal grafts, and applications of gibberellin (GA) and indoleacetic acid (IAA) were used to localize the site of control for stem elongation in cucumber (Cucumis sativus L.). Dwarf and tall plants were reciprocally grafted to determine influence of stems and roots on stem elongation. At 21 days there were no significant differences in length between stems grafted to their own roots and those grafted to roots of the other type. GA(3), GA(4+7), and IAA were applied to seedlings with and without live apical buds. Seedlings with live apical buds responded to level of added GA, but not to added IAA. GA(4+7) was more effective than GA(3). Hypocotyls of tall plants responded more to both GA treatments than did those of the dwarves when both types had live apical buds. When either GA(4+7) or IAA was applied to seedlings with dead apical buds, elongation of the hypocotyl responded to level of the growth regulator, but there was no difference in response between the dwarf and tall plants.

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Assay and Biochemical Properties of the Proteinase Inhibitor-inducing Factor, a Wound Hormone.

An assay has been developed for the proteinase inhibitor-inducing factor (PIIF), a wound hormone. PIIF is present in tomato (Lycopersicum esculentum var. Bonnie Best) leaf extracts and induces accumulation of proteinase Inhibitor I when the extracts are supplied briefly to excised leaves that are subsequently incubated in water under constant light. An active water-soluble crude PIIF solution was conveniently prepared from autoclaved and lyophilized tomato leaves. Accumulation of Inhibitor I, induced by crude PIIF, is linear, commencing at about 8 to 10 hours after feeding and continues for several hours. Evidence is presented that the PIIF-induced accumulation of Inhibitor I, determined immunologically, is accompanied by the accumulation of other trypsin and chymotrypsin inhibitors, determined enzymatically. The accumulation of Inhibitor I is inhibited by actinomycin D and cycloheximide but not by chloramphenicol or rifampin. PIIF cannot be replaced by traumatin, indoleacetic acid, gibberellic acid, kinetin, ethylene, or abscisic acid. PIIF activity was not destroyed by incubation with a number of proteolytic, carbohydrase, phosphatase, or pyrophosphatase enzymes. The active substance is insoluble in lipid solvents.

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Multiple amine oxidases in cucumber seedlings.

Cell-free extracts of cucumber (Cucumis sativus L. cv. National Pickling) seedlings were found to have amine oxidase activity when assayed with tryptamine as a substrate. Studies of the effect of lowered pH on the extract indicated that this activity was heterogeneous, and three amine oxidases could be separated by ion exchange chromatography. The partially purified enzymes were tested for their activities with several substrates and for their sensitivities to various amine oxidase inhibitors. One of the enzymes may be a monoamine oxidase, although it is inhibited by some diamine oxidase inhibitors. The other two enzymes have properties more characteristic of the diamine oxidases. The possible relationship of the amine oxidases to indoleacetic acid biosynthesis in cucumber seedlings is discussed.

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Auxin and the response of pea roots to auxin transport inhibitors: morphactin.

The auxin transport inhibitor methyl-2-chloro-9-hydroxyfluorene-9-carboxylate (CFM), a morphactin, inhibits negative geotropism, causes cellular swelling, and induces root hair formation in roots of intact Pisum sativum L. seedlings. In excised pea root tips, CFM inhibits elongation more than increase in fresh weight (swell ratio = 1.3 at 20 mum CFM). CFM growth inhibition was expressed in the presence of ethylene. Indoleacetic acid (IAA) prevented the expression of CFM growth inhibition possibly because IAA inhibited the accumulation of CFM into the tissue sections. CFM inhibited the accumulation of IAA and 2,4-dichlorophenoxyacetic acid into excised root tips. Applying Leopold's (1963. Brookhaven Symp. Biol. 16: 218-234) model for polar auxin transport, this result suggests a possible explanation for CFM inhibition of geotropism in pea roots, i.e. disruption of auxin transport by interfering with auxin binding.

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C(2)H(4): Its Incorporation and Metabolism by Pea Seedlings under Aseptic Conditions.

The effects of various treatments on the recently reported system in pea (Pisum sativum cv. Alaska), which results in (a) the incorporation of (14)C(2)H(4) into the tissue and (b) the conversion of (14)C(2)H(4) to (14)CO(2), was investigated using 2-day-old etiolated seedlings which exhibit a maximum response. Heat treatment (80 C, 1 min) completely inhibited both a and b, whereas homogenization completely inhibited b but only partially inhibited a. Detaching the cotyledons from the root-shoot axis immediately before exposing the detached cotyledons together with the root-shoot axis to (14)C(2)H(4) markedly reduced both a and b. Increasing the (14)C(2)H(4) concentration from 0.14 to over 100 mul/l progressively increased the rate of a and b with tissue incorporation being greater than (14)C(2)H(4) to (14)CO(2) conversion only below 0.3 mul/l (14)C(2)H(4). Reduction of the O(2) concentration reduced both a and b, with over 99% inhibition occurring under anaerobic conditions. The addition of CO(2) (5%) severely inhibited (14)C(2)H(4) to (14)CO(2) conversion without significantly affecting tissue incorporation. Exposure of etiolated seedlings to fluorescent light during (14)C(2)H(4) treatment was without effect. Similarly, indoleacetic acid, gibberellic acid, benzyladenine, abscisic acid, and dibutyryl cyclic adenosine monophosphate had no significant effect on either a or b.The possibilities that the incorporation of (14)C(2)H(4) into pea tissues and its conversion to (14)CO(2) is linked to ethylene action, or that it represents a means of reducing the endogenous ethylene level, are discussed.Several problems encountered with the use of polyethylene vials, rubber serum stoppers, Clorox, and microbial contamination are also described.

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Auxin transport inhibitors: fluorescein and related compounds.

Fluoresceins are shown to be effective inhibitors of indoleacetic acid transport as measured by the receiver agar block technique, eosin having the same order of activity as 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid, with fluorescein less effective. It is suggested that many of their characteristic effects on plants, especially those which involve auxin, are at least partially due to their effects on auxin transport.

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Auxin: its role in genetic tumor induction.

Seedlings of the tumor-prone amphiploid Nicotiana suaveolens X N. langsdorffii were grown on nutrient medium supplemented with indoleacetic acid (IAA) and scored at regular intervals for the incidence of tumor formation. IAA at 2 x 10(-5)m caused a significant reduction in the rate of tumor formation. Plants were also grown on nutrient medium under two different intensities of illumination, and the endogenous level of IAA was determined in 23-day-old seedlings. Those grown under 2000 ft-c of illumination had a higher incidence of tumors and a significantly lower level of endogenous IAA than those grown under 500 ft-c of illumination. A correlation in time between decline in the endogenous level of IAA and onset of tumor formation was demonstrated in greenhouse-grown plants.

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