Inhibition of Auxin-induced Deoxyribonucleic Acid Synthesis and Chromatin Activity by 5-Fluorodeoxyuridine in Soybean Hypocotyl

Rootless soybean (Glycine max) seedlings were used as a test system to examine the action of auxin on chromatin-directed RNA synthesis. Chromatin from the basal tissue of rootless seedlings (both control and auxin-treated) had RNA synthetic capacity similar to that of chromatin from comparably treated intact seedlings. When DNA synthesis normally induced in the basal tissue by auxin was blocked in the rootless seedlings by 5-fluorodeoxyuridine, the auxin enhancement of chromatin activity was inhibited 70%. This level was still three times the control level, indicating that auxin influenced the synthetic activity of existing DNA template. Experiments with Escherichia coli RNA polymerase revealed that chromatin from both auxin- and auxin plus 5-fluorodeoxyuridine-treated tissue saturated at higher levels than chromatin from control tissue.     

Auxin-induced enhancement of RNA polymerase activity in soybean as a function of development

Studies on chromatin and solubilized RNA polymerase from control and 2,4-D treated whole hypocotyls indicate that the activity of RNA polymerase I is enhanced by the auxin treatment while the activity of polymerase II is essentially the same in control and treated tissue. However, different portions of the hypcotyl respond differently to the auxin treatment. The enhancement of solubilized polymerase I activity is greater in the lower half of the hypocotyl than it is in the upper half. In the hook region an inhibition of RNA solubilized polymerase activity is observed. Similarly, 2,4-D treatment results in an inhibition of chromatin bound RNA polymerase activity in nuclei isolated from first leaves. Chromatography on DEAE-Sephadex also reveals a selective enhancement of polymerase I in the upper and lower halves of the hypocotyl.     

Photoaffinity-labeled Auxins: Synthesis and Biological Activity

Two light-sensitive analogs of 2,4-dichlorophenoxyacetic acid, namely 4-azido-2-chlorophenoxyacetic acid and 3-azido-5-chlorophenoxyacetic acid, have been synthesized for use as auxin photoaffinity labels. The preparation and biological activity of the compounds are described. Both contain the photolabile azido group; the 2,4-substituted compound shows auxin activity and the 3,5-substituted compound does not. These photoaffinity analogs of 2,4-dichlorophenoxyacetic acid may be useful in the identification of the auxin receptor molecules in plant cells and eventually of the receptor sites within these molecules.     

The relative amounts and identification of some 2,4-dichlorophenoxyacetic Acid metabolites isolated from soybean cotyledon callus cultures

Soybean (Glycine max L.) cotyledon callus grown on radioactive 2,4-dichlorophenoxyacetic acid (2,4-D-1-¹⁴C) as an auxin produced 2,4-D metabolites, which qualitatively and quantitatively changed with time. Water soluble fractions from the tissue exhibited a steady increase in radioactivity during the course of 24 days. Following β-glucosidase treatment, at least eight aglycones were obtained from the water soluble fraction of the tissue after 8 days. The metabolite, 4-hydroxy-2,5-dichlorophenoxyacetic acid was the most abundant aglycone during the entire 32 day growth period while 4-hydroxy-2,3-dichlorophenoxyacetic acid was detected as a minor metabolite. Radioactivity in the ether soluble acidic fractions reached a maximum of 82% of the total in the tissue after 2 days. The level then decreased to 44% by the end of 24 days. A total of seven ether soluble components were detected. In addition to 2,4-D glutamic acid, which was detected in high amounts after 24 hours, 2,4-D aspartic acid was found to be the most abundant ether soluble metabolite after longer time periods. Mass spectral data and a fragmentation pattern are presented for 2,4-D aspartic acid.     

Auxin-induced changes in the population of translatable messenger RNA in elongating sections of soybean hypocotyl

In vitro translation products of polyadenylated RNA from untreated and auxin-treated elongating sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. The levels of translatable messenger RNA for at least ten in vitro translation products are increased by auxin treatment. The induction by auxin occurs rapidly (within 15 minutes), and the amounts of the induced in vitro translation products increase with time of auxin treatment. Indoleacetic acid has the same effect on the population of translatable messenger RNA as 2,4-dichlorophenoxyacetic acid. The auxin-induced in vitro translation products disappear rapidly when Actinomycin D is present during the last two hours of a three-hour auxin treatment.     

2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells

The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 micrograms/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture (greater than 5 micrograms/ml).     

Auxin- and ethylene-induced changes in the population of translatable messenger RNA in Basal sections and intact soybean hypocotyl

In vitro translation products of polyadenylated RNA from untreated and auxin-treated basal sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. Within one hour of 2,4-dichlorophenoxyacetic acid treatment, the translatable messenger RNAs for at least twelve in vitro translation products are modulated upward. In vitro translation products of polyadenylated RNA from untreated, auxin-treated and Ethephon-treated intact soybean hypocotyl were also analyzed. Within two hours of treatment with either 2,4-dichlorophenoxyacetic acid or Ethephon, the translatable messenger RNAs for a group of high molecular weight in vitro translation products are modulated upward. There is a particular set of translatable messenger RNA, encoding in vitro translation products in the 24,000 to 32,000 molecular weight range, that is specifically modulated upward by auxin treatment in intact soybean hypocotyl and in hypocotyl sections.     

Auxins induce alpha-amylase activity in pea cotyledons

The report documents how the development of α-amylase activity in detached cotyledons of Pisum sativum cv Alaska is accelerated 2- to 12-fold during incubation with 1 micromolar to 10 micromolar 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, or with 4-chloroindoleacetic acid, an endogenous auxin from Pisum sativum. It seems probable that auxin from the embryonic axis induces α-amylase in the attached cotyledons during germination.     

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A₇ (GA₇), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA₇ also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA₇ solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.     

Oxidation of NADH by hypocotyl segments from soybean is stimulated by 2,4-D

Sections cut from regions of cell elongation of hypocotyls of dark-grown soybean seedlings oxidized externally supplied NADH as estimated from the decrease in A₃₄₀ measured spectrophotometrically. The oxidation of NADH by 1-cm sections was stimulated 1.5- to 2-fold by 1 μM of the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D-Stimulated oxidation of NADH was resistant to cyanide. Stimulations were also given by the naturally occurring auxin, indole-3-acetic acid (IAA) but not by the growth inactive 2,4-D analog 2,3-dichlorophenoxyacetic acid (2,3-D) and the growth inactive β-naphthaleneacetic acid (β-NAA). Since NADH is a membrane impermeant substrate, the findings confirm studies with inside-out and right-side-out vesicles that show the 2,4-D-stimulated NADH oxidase to be located at the external cell surface. Cut surfaces are not responsible for the activity as shown by experiments with lanolin-sealed sections. The external NADH oxidase measurements do not require special equipment and exhibit characteristics normally associated with enzyme-catalyzed reactions.     

Quantitative Effects of 2,4-Dichlorophenoxyacetic Acid on Growth of Suspension-cultured Acer pseudoplatanus Cells

Using suspension-cultured Acer pseudoplatanus cells requiring 2,4-dichlorophenoxyacetic acid for growth, the dependence of the population doubling time and the maximum increase in cell population density on the auxin concentration was studied. It appears that in the range of 2,4-dichlorophenoxyacetic acid concentration from 4 × 10⁻⁸ to 4 × 10⁻⁶ M, the rate of cell division during the logarithmic growth phase is independent of the auxin concentration, while the maximum number of cell generations obtained is limited by the initial auxin concentration. The significance of these two aspects of auxin action are discussed.     

Effect of 2,4-dichlorophenoxyacetic acid on polysomal profiles and polyadenylated RNA in excised tuber tissue of Jerusalem artichoke (Helianthus tuberosus)

Dormant tuber tissue of Jerusalem artichoke ( Helianthus tuberosus L.) can be stimulated by wounding to initiate RNA and protein synthesis. No DNA synthesis or cell divisions occur unless an auxin is provided. Changes in polysomal profiles and levels of Poly(A)⁺-RNA in response to wounding and auxin treatment were studied. Polysomes were isolated at various times after excision and incubation of tissue in the presence or absence of 10⁻⁵ M 2,4-dichlorophenoxyacetic acid. Polysomal profiles were studied by sucrose density gradient centrifugation. Dormant tissue contained ribosomes mainly in monosome form. Within 4 h of excision, a significant increase in the polysomal fraction was observed both in control and auxin-treated tissue. Increases in polysomes continued during the next 20 h. Poly(A)⁺-RNA was isolated from total polysomal RNA by oligo(dT)-cellulose column chromatography. There was a large increase in the amount of poly(A)⁺-RNA within 4 h of excision. During the first 43 h of incubation, levels of total polysomal RNA as well as poly(A)⁺-RNA in tissue treated with 2,4-dichlorophenoxyacetic acid were significantly higher than those in controls.     

Auxin-induced synthesis of TB-RNA in pea stems

The effect of 2,4-dichlorophenoxyacetic acid on RNA synthesisin etiolated pea internode segments was studied. Auxin stimulatedsynthesis of TB-RNA only in short period incubation, suggestingits primary importance in auxin action. The nucleotide compositionof newly synthesized TB-RNA seems to be modified under auxininfluence. (Received February 8, 1969; )     

Stimulation of ATPase activity by auxin is dependent on ATP concentration

The effect of auxin on membrane-bound ATPase activity was studied in a plasma membrane-enriched fraction from zucchini hypocotyls. The apparent K_M of ATPase activity for ATP was decreased in the presence of 10^-6 M auxin so that at very low ATP concentrations the stimulation of ATPase activity was most obvious. The weak auxin analogue, 2,3-dichlorophenoxyacetic acid, stimulated much less than the active auxin 2,4-dichlorophenoxyacetic acid.     

2,4-Dichlorophenoxyacetic Acid-enhanced Phosphorylation of Soybean Nuclear Proteins

In vitro nuclear protein phosphorylation is enhanced in nuclei isolated from 2,4-dichlorophenoxyacetic acid (2,4-d)-treated mature soybean (Glycine max) hypocotyl relative to nuclei from untreated tissue. Increased nuclear protein phosphorylation correlates with increased levels of nuclear protein kinase activity. These changes generally parallel previously reported 2,4-d-enhanced RNA polymerase activity of these nuclei and the in vivo levels of RNA synthesis. Phosphate incorporation represents bona fide protein phosphorylation, with 87% of the label being identified as phosphoserine and 7% as phosphothreonine. Label from [γ-³²P]adenosine 5′-triphosphate is incorporated primarily into various nonhistone fractions with the greatest accumulation in loosely associated fractions (either released during incubation with ATP or removed by 0.15 m Nacl). Although electrophoretic analysis on sodium dodecyl sulfate gels shows no differences in the protein profiles of the loosely associated or sodium dodecyl sulfate-soluble nonhistone proteins, there are changes in the pattern of phosphorylation of other proteins, after 2,4-d treatment. Acid-soluble basic nuclear proteins are phosphorylated to a much lower extent than are the other nuclear protein fractions. While histone F₁ is subject to slight phosphorylation when nuclei are labeled in vitro, phosphorylation of the other histones is undetectable. One acid-soluble protein shows a substantial increase in quantity and in phosphorylation after 2,4-d treatment. This protein is similar in electrophoretic mobility to pea histone F₁ but its identity is unknown. Urea-acetic acid gels of the acid-soluble nuclear proteins show that auxin treatment results in increased quantities and in increased phosphorylation of various low mobility nonhistone basic nuclear proteins.     

EFFECT OF AUXIN AND ANTIAUXIN ON THE GROWTH AND RNA SYNTHESIS OF ETIOLATED PEA INTERNODE

Using etiolated Alaska pea internode segments the followingresults were obtained. An auxin, 2,4-dichlorophenoxyacetic acid, 1 mg/liter, inducedthe elongation growth of the segment and stimulated the biosynthesisof RNA, particularly of messengar RNA, in 3 hr incubation. Thesimilar stimulation of RNA synthesis and of elongation due toindole-3-acetic acid was found in 1 hr incubation. The stimulativeeffect of 2,4-dichlorophenoxyacetic acid on the elongation andon the synthesis of messenger RNA was inhibited by the additionof an antiauxin, transcinnamic acid. Significance of the auxin-inducedsynthesis of messenger RNA in producing protein responsiblefor the stress relaxation of cell wall is discussed. (Received June 13, 1967; )     

Plasma membrane protein composition and synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary The types and amount of plasma membrane proteins synthesized during cell elongation in response to auxin (2,4-dichlorophenoxyacetic acid) treatment were investigated. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [³⁵S]methionine for various times, ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. Protein accumulated in the plasma membrane after auxin treatment. Despite this accumulation, the protein incorporation rate, estimated by the amount of label in the plasma membrane following a 0.5 h [³⁵S]methionine pulse, was unaffected by auxin treatment at both 0.5 and 18 h of treatment. Protein apparently accumulated by a mechanism distinct from enhanced incorporation. The plasma membrane proteins synthesized by elongating segments differed from controls at 18 h, as evidenced by the pattern of fluorographs following a 0.5 h radiolabelling. However, auxin treatment did not alter the 2-D gel pattern of the polypeptides detectable by silver stain.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IEF isoelectric focusing - PM plasma membrane - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The Influence of Auxin and Ethylene on Chromatin-directed Ribonucleic Acid Synthesis in Soybean Hypocotyl

Soybean seedlings treated with ethylene exhibited small increases in ribonucleic acid content in the elongating section of the hypocotyl. Chromatin isolated from the elongating section of ethylene-treated seedlings showed a 35 to 60% increase in the capacity for RNA synthesis. The ethylene-induced response was saturated at 1 microliter/liter of ethylene and was fully expressed after 3 hours. Auxin caused marked accumulation of RNA and DNA in the elongating and basal tissue of the hypocotyl. Chromatin isolated from these auxin-treated tissues showed an 8- to 10- fold increase in RNA synthetic capacity as measured in vitro. Ethylene added with auxin reduced the auxin enhancement of nucleic acid synthesis in the elongating and basal tissues. Both ethylene and auxin treatment of the seedlings inhibited nucleic acid accumulation and chromatin activity in the apical tissue. Ethylene did not appear to mediate the auxin effects on nucleic acid synthesis in soybean hypocotyl with the possible exception of inhibition in the apical tissue.