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.     

Greater Length of Ribonucleic Acid Synthesized by Chromatin-bound Polymerase from Auxin-treated Soybean Hypocotyls

An investigation has been made of the RNA synthesized by chromatin-bound RNA polymerase from soybean hypocotyls (Glycine max var. Wayne). Polymerase activity is 4- to 5-fold higher with chromatin from tissue treated with 2,4-dichlorophenoxyacetic acid, a synthetic auxin, compared to untreated tissue. Thin layer chromatography of the RNA hydrolysis products and acrylamide gel electrophoresis of the RNA synthesized by the chromatin show that increased activity induced by 2,4-dichlorophenoxyacetic acid is due primarily to the production of longer RNA chains, with only 20 to 50% increase in the number of RNA chains. The observation that 2,4-dichlorophenoxyacetic acid treatment leads to greater rates of RNA synthesis, producing longer chains in unit time, suggests that one manifestation of auxin activity is in activation of RNA polymerase I (ribosomal RNA polymerase).     

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.     

SENESCENCE: HORMONAL CONTROL OF RNA AND PROTEIN SYNTHESIS IN EXCISED BEAN POD TISSUE

The kinetics of degradation of RNA, total protein, ribosomal and soluble (R-S) protein and DNA were followed over 24 hours in excised segments of Kentucky Wonder pole beans (Phaseolus vulgaris). In absence of exogenous auxin, RNA degradation proceeded from about zero time. By 15 hours, after about 20% of the RNA had degraded, the degradation of DNA, total protein and R-S protein was initiated. Exogenous auxin (25 ppm α-napthaleneacetic acid) prevented these degradations. The addition of kinetin had little or no effect. Auxin enhanced incorporation of orotic acid into RNA two to several times more than it did amino acids into protein. Actinomycin D repressed RNA synthesis about 75% including a repression of auxin-induced synthesis. It had no effect on the basal level of protein synthesis but did repress auxin-induced synthesis. The results indicate that the primary action of auxin in preventing senescence of bean endocarp tissue is at the site of RNA synthesis, and the effect of auxin on DNA and protein is a consequence of the effect of auxin on RNA metabolism.     

CHANGES IN CHROMATIN TEMPLATE ACTIVITY AND THEIR RELATIONSHIP TO DNA SYNTHESIS IN MOUSE PAROTID GLANDS STIMULATED BY ISOPROTERENOL

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-³H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.     

Comparative studies of effect of auxin and ethylene on permeability and synthesis of RNA and protein

The effects of ethylene on permeability and RNA and protein synthesis were assayed over a 6 to 26 hr period in tissue sections from avocado (Persea gratissima Gaertn. F., var. Fuerte), both pulp and peel of banana (Musa sapientum L., var. Gros Michel), bean endocarp (Phaseolus vulgaris L., var. Kentucky Wonder Pole beans) and leaves of Rhoeo discolor. Ethylene had no effect on permeability in 4 of the 5 tissues, but sometimes enhanced solute uptake in banana peel; it had either no effect or an inhibitory effect on synthesis of RNA and protein in sections from fruits of avocado and banana. Auxin (α-naphthalene acetic acid) stimulated synthesis of RNA and protein in bean endocarp and Rhoeo leaf sections, whereas ethylene inhibited both basal and auxin-induced synthesis. It is concluded that in these tissues the auxin effect is not an ethylene effect.     

Fractionation of nucleoli from auxin-treated soybean hypocotyl into nucleolar chromatin and preribosomal particles

Nucleoli from auxin-treated tissues (Glycine max L. var Wayne or Kaoshiung No. 3) were isolated and purified by Percoll density gradient centrifugation. There was a 2.1-fold increase in RNA and a 2.8-fold increase in protein after a 24-h auxin treatment per unit nucleolar DNA. More than 150 acid-soluble protein spots were associated with the auxin-treated nucleoli on two dimensional (2-D) gel electropherograms.

Nucleoli from auxin-treated tissue were fractionated by suspension in 20 millimolar dithiothreitol at room temperature for 20 minutes into two distinct fractions referred to as the nucleolar chromatin and preribosomal particle fractions. The DNA:RNA:protein ratio of the chromatin fraction was 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNA recovered in this fraction. The acid-soluble proteins in the chromatin were resolved into 32 protein spots on 2-D gel electropherogram. The most abundant spots were identified as histones.

The nucleolar preribosomal particle fraction had a DNA:RNA:protein ratio of 1:24:102 and contained only trace amounts of RNA polymerase 1 activity and only 10 per cent of the nucleolar DNA. Acid-soluble proteins associated with these particles were resolved into 78 protein spots; 72 of these (acid-soluble) protein spots corresponded in 2-D gel electrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 protein spots found in 80S ribosomal proteins were absent in the preribosomal particles. It seems reasonable, based on these data, that the enlargement of nucleoli after auxin treatment is primarily due to the large increase in ribosomal proteins and rRNA which accumulate and assemble in the nucleoli in the form of preribosomal particles.

     

Chromatin-directed Ribonucleic Acid Synthesis: A Comparison of Chromatins Isolated from Healthy, Avirulent Agrobacterium tumefaciens Inoculated, and Crown-Gall Tumor Tissues of Vicia faba

Chromatin was extracted from healthy, avirulent Agrobacterium tumefaciens inoculated, and crown-gall tumor Vicia faba internodes of the same age. Chromatin from crown-gall tissue produced 5 times more RNA per 100 micrograms of DNA than chromatin from the healthy tissue. When template availability was compared using chromatin with saturating amounts of Escherichia coli RNA polymerase, chromatin from crown-gall tissue had 36% more available template than the controls. In addition, when γ-³²P-ATP was incorporated into the RNA synthesizing reaction mixture, with saturating amounts of E. coli RNA polymerase, there were twice as many RNA chain starts in tumor as in control tissue.     

RNA Stimulated by Indole Acetic Acid

THE stimulation of RNA synthesis in plant cells by auxins^1,2 may be due, in part, to gene derepression, since chromatin isolated from hormone-treated cells is a better template. Mathysse and Phillips³ demonstrated that an acceptor protein for auxin can interact with the chromatin to derepress the genome. In their system the hormone and protein do not affect the rate of RNA synthesis if pure DNA. is used as a template. However, the actual mechanism of auxin on RNA synthesis by isolated RNA polymerase system has yet to be elucidated.     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.     

URIDINE-5-H3 RADIOAUTOGRAPHY OF THE HUMAN SEX CHROMATIN BODY

To obtain an estimate of the rate of RNA synthesis by the heterochromatic sex chromatin body, human female fibroblasts were labeled with uridine-5-H³ and radioautographed. The number of grains over the sex chromatin body was compared with the number of grains over a comparable area of euchromatin. The ratio was 0.37. When corrected for the higher content of DNA per unit area in heterochromatin, the maximum rate of RNA synthesis by the DNA of the sex chromatin body was approximately 18% of the rate of RNA synthesis by a comparable amount of euchromatin DNA. The rate of RNA synthesis by the sex chromatin body did not increase significantly with partial despiralization of this chromatin at prophase.     

Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal regulation of plant growth

Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day. This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs. Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme. The inhibition was observed within 30 min of the hormone application, and maximal effect—90% inhibition—after 18 hours. Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 0.12 microliters per liter ethylene and maximal response at 1.2 microliters per liter. Ethylene provoked a 5-fold increase in the K_m^app of arginine decarboxylase for its substrate and reduced the V_max^app by 10-fold. However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere. Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings. On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed.     

Induction of DNA synthesis by 2,4-dichlorophenoxyacetic acid during callus induction in Jerusalem artichoke tuber tissues

During the induction of DNA synthesis in Jerusalem artichoke (Helianthus tuberosus L.) tuber by 2,4-D, the 2-¹⁴C-2, 4-D from the agar medium rapidly incorporated into the ethanol soluble and insoluble fractions. Although the 2,4-D level in the ethanol soluble fraction decreased on transplantation of the tissue from the 2-¹⁴C-2,4-D medium to medium without the auxin, its level in the buffer-soluble and -insoluble macromolecular fractions increased. The purified, buffer-insoluble macromolecules were chromatin. The 2,4-D binding to chromatin particularly increased during DNA synthesis. The histone contents of chromatin decreased as DNA synthesis progressed. The polyacrylamide gel electrophoretic patterns of the histones showed a decrease in the moderately lysine-rich histone fraction as compared to other fractions. Thus, the decrease in the histone level caused by 2,4-D and the presence of the 2,4-D moderately lysine-rich histone complex may be closely related to the induction of DNA synthesis by 2,4-D in cells.     

Template activity of chromatin during stimulation of cellular proliferation in human diploid fibroblasts

1. Contact-inhibited confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by replacing the medium with fresh medium containing 30% foetal calf serum. 2. Of the cells 40–75% are stimulated to divide with a peak DNA synthesis between 15 and 21h and a peak mitotic index between 28 and 30h after stimulation. 3. In the first 12h before the initiation of DNA synthesis there is a biphasic increase in the incorporation of [³H]uridine into RNA of whole cells. 4. This is paralleled by a similar biphasic stimulation of chromatin template activity measured in vitro in a system in which purified cell chromatin is incubated with an exogenous RNA polymerase isolated from Escherichia coli. 5. The changes in chromatin template activity are believed to represent activation of the genome, with more sites available for RNA synthesis, and to account almost entirely for the changes in RNA synthesis occurring in the whole cell.     

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.     

Hormonal control of deoxyribonucleic Acid and protein syntheses in pea root cortical explants

The hormonal control of DNA and protein syntheses in cortical explants taken at 10 to 11 mm from the tip of 3-day-old seedling roots (Pisum sativum cv. Little Marvel) was examined. On the auxin medium, S2M, the cortical cells began to enlarge at day 4 in culture, with no DNA synthesis or cell division throughout the 7-day culture period. With the addition of kinetin to this medium, S2M + K, the DNA content of the explants increased about three times by day 3, with further increases thereafter. This DNA increase was followed by cell division activity and subsequent tracheary element differentiation initiated at day 5. At least two divisions per parent cortical cell were required prior to this cytodifferentiation. The absolute hormonal requirements for the DNA synthesis and cell division responses were substantiated by the lack of either response in explants cultured on basal (S2M medium minus auxins) or basal + K medium for 7 days. On the auxin medium, there was no protein accumulation in the cortical explants over the 7-day period. On S2M + K medium, protein accumulation began after day 2 with a steady rate of increase until day 4, and some fluctuation thereafter. The pattern of increasing uptake of ¹⁴C-leucine was similar for days 0 to 4 in explants on either medium. After day 4 on S2M, the uptake continued to increase coincident with cell enlargement initiation, whereas on S2M + K there was a decline. Incorporation of ¹⁴C-leucine into trichloroacetic acid-precipitates of the total buffered homogenate from explants on both media exhibited a similar pattern, i.e. an increase during days 0 to 3 and then a decline to a level about three times higher than day 0. Incorporation into the homogenate soluble fraction also showed a similar pattern in explants cultured with or without kinetin. From the differences in net protein accumulation and the incorporation data, speculation on a cytokinin effect on protein synthesis and degradation rates is presented.