DNA synthesis in the elongating nondividing cells of the lentil epicotyl and its promotion by gibberellin

Two-day-old lentil seedlings, (Lens culinaris Med.) were incubated for a 48-hour period with and without gibberellin (GA) in the presence and absence of 5-fluorodeoxyuridine (FUDR). The number of cells per epicotyl did not increase during this period. Growth of the epicotyl was thus due to cell elongation alone.

The elongating cells of this tissue synthesized DNA. GA promoted and FUDR inhibited cell elongation, DNA synthesis, and RNA synthesis in the tissue.

FUDR promoted uptake of thymidine and thymidine incorporation into cellular DNA, presumably by inhibiting synthesis of endogenous thymidine. Presence of GA promoted thymidine incorporation into cellular DNA and uridine incorporation into cellular RNA. In either case, there was no effect on the uptake of the precursor into the tissue.

Fractionation of thymidine-labeled nucleic acids on a MAK column showed that thymidine was exclusively incorporated into the DNA fraction. Presence of GA promoted thymidine incorporation into this fraction and also increased the amount of ribosomal RNA.

The data provide direct evidence for the conclusion that DNA synthesis is necessary for elongation of certain plant cells.

     

Regulation of Synthesis of Two Immunologically Distinct Nucleic Acid-Dependent Nucleoside Triphosphate Phosphohydrolases in Vaccinia Virus-Infected HeLa Cells

The two nucleic acid-dependent nucleoside triphosphate phosphohydrolases, previously purified from vaccinia virus cores, were shown to be immunologically distinct enzymes. Antiserum prepared against purified phosphohydrolase I and antiserum prepared against purified phosphohydrolase II only neutralized the activity of that enzyme used as antigen. Both enzymes were induced in HeLa cells after vaccinia infection. DNA-cellulose chromatography was used to purify the two phosphohydrolases from the cytoplasms of infected cells. The enzymes were identified by their different substrate specificities, nucleic acid dependence, and neutralization with specific antiserum. A third chromatographically separable nucleic acid-dependent phosphohydrolase similar to phosphohydrolase I in substrate specificity but not neutralizable by antiserum to either phosphohydrolase I or II, was also isolated from infected cells. No nucleic acid-dependent nucleoside triphosphate phosphohydrolase activity was detected by similar methods from uninfected HeLa cells. Formation of these virus-induced enzymes was prevented by actinomycin D and cycloheximide, indicating a requirement for de novo RNA and protein synthesis, respectively. The kinetics of induction and inhibition by cytosine arabinoside, an inhibitor of DNA synthesis, suggested that synthesis of the phosphohydrolases is a late viral function. Rifampin, an inhibitor of vaccinia virus growth which prevents virion assembly, had no inhibitory effect on the induction of the phosphohydrolases. This result was consistent with the finding that these enzymes exist in a soluble as well as in a particulate form in the cytoplasm of infected cells. Addition of another specific anti-poxviral drug, isatin-beta-thiosemicarbazone, to vaccinia-infected cells partially inhibited induction of the phosphohydrolases.     

Changes in RNA levels in the shoot apex of Silene during the transition to flowering

Changes in RNA concentration in the shoot apical meristem during induction and the transition to flowering were measured histochemically in Silene coeli-rosa (L.) Godron, a long-day plant. In the apices of plants induced by 7 long days the RNA concentration increased to about 25 per cent higher than in non-induced plants. Three long days did not induce flowering but resulted in a transient rise in RNA concentration. When plants were given long days interrupted by varying numbers of short days successful induction was accompanied by a sustained increase in RNA concentration but those treatments which were not inductive gave only transient increases in RNA. Gibberellic acid had no effect on induction or apical growth rates but increased the RNA concentration by 50 per cent or more in both induced and non-induced plants. Plants induced to flower at 13° C had the same RNA concentration and growth rate at the apex as in non-induced plants at 20° C. Since changes in RNA concentration in the apex could occur without changes in growth rate and without flowering, and induction could occur without a change in RNA concentration or growth rate, it is suggested that the increase in RNA and growth rate which normally occur at the transition to flowering might not be essential for the formation of a flower but may be more closely related to the rapid growth associated with the formation of the inflorescence.Abbreviations LD long day - SD short-day     

Treatments that Enhance Flowering in the Post-inductive Period of a Short-day Plant, Chenopodium rubrum L.

Flowering was promoted in young plants of Chenopodium rubrumL. by application of growth inhibitors such as 5-fluorodeoxyuridine(FUDR) and (2-chloroethyl) trimethylammonium chloride (CCC),growth substances (indol-3yl-acetic acid, IAA), by the removalof roots and by drought. All the treatments were effective onlyduring the post-inductive period and at the threshold levelof photoperiodic induction. The response of plants was strictlytime-dependent. The experimental data indicate that the stimulationof flowering is usually accompanied by inhibition of leaf initiationand growth. The treatments probably produced variation in thequantitative expression of flowering by causing a shift in emphasisin the development of leaf and bud primordia at the shoot apex.The dynamic analysis of differentiation of the shoot apex indicatesa correlation between the morphological stage of the shoot apexand its responsiveness to the treatments.     

Ethylene and ABA interactions in the regulation of flower induction in Pharbitis nil

Hormones are included in the essential elements that control the induction of flowering. Ethylene is thought to be a strong inhibitor of flowering in short day plants (SDPs), whereas the involvement of abscisic acid (ABA) in the regulation of flowering of plants is not well understood. The dual role of ABA in the photoperiodic flower induction of the SDP Pharbitis nil and the interaction between ABA and ethylene were examined in the present experiments. Application of ABA on the cotyledons during the inductive 16-h-long night inhibited flowering. However, ABA application on the cotyledons or the shoot apices during the subinductive 12-h-long night resulted in slight stimulation of flowering. Application of ABA also resulted in enhanced ethylene production. Whereas nordihydroguaiaretic acid (NDGA) - an ABA biosynthesis inhibitor - applied on the cotyledons of 5-d-old seedlings during the inductive night inhibited both the formation of axillary and of terminal flower buds, application of 2-aminoethoxyvinylglycine (AVG) and 2,5-norbornadiene (NBD) - inhibitors of ethylene action - reversed the inhibitory effect of ABA on flowering. ABA levels in the cotyledons of seedlings exposed to a 16-h-long inductive night markedly increased. Such an effect was not observed when the inductive night was interrupted with a 15-min-long red light pulse or when seedlings were treated at the same time with gaseous ethylene during the dark period. Lower levels of ABA were observed in seedlings treated with NDGA during the inductive night. These results may suggest that ABA plays an important role in the photoperiodic induction of flowering in P. nil seedlings, and that the inhibitory effect of ethylene on P. nil flowering inhibition may depend on its influence on the ABA level. A reversal of the inhibitory effect of ethylene on flower induction through a simultaneous treatment of induced seedlings with both ethylene and ABA strongly supports this hypothesis.     

Rhythmic changes in ribonuclease activity in relation to nucleic acid synthesis in cotyledons ofChenopodium rubrum L. and to floral induction of the plants

Ribonuclease (RNAse) activity was investigated in cotyledons ofChenopodium rubrum plants subjected to various conditions of illumination (photoperiodic induction, continuous light, induction cancelled by interrupting the dark period by a light-break). At the end of the dark period of the single inductive cycles RNAse activity of induced plants was inferior to that of plants grown in continuous light. At the end of the first two cycles the activity was lowest after the interruption of the dark period by light. The investigation of the enzyme in 6h intervals showed rhythmic changes in activity to occur in induced plants. Enzyme activity followed a pattern opposed to this of nucleic acid (NA) synthesis in the cotyledons. In plants from continuous light the enzyme activity did not show any rhythm and in plants having obtained a light-break during the inductive period the rhythm was less distinct than in the induced ones. The period length of the endogenous rhythm of NA synthesis in the cotyledons is about half as long as this of flowering and the peaks of flowering coincide with the throughs of NA synthesis.     

The Negative Response of Photoperiodic Floral Induction in Chenopodium rubrum L. to Preceding Growth

In Chenopodium rubrum there exists a correlation between theage of the seedlings and the effectiveness of photoperiodicinduction. The younger the plants the more effective was photoperiodictreatment. In three-day-old seedlings one short day was sufficientto promote incomplete flowering, while two short days broughtabout 100 per cent flowering. With six-, eight-, and ten-day-oldplants exposed to two or three short days quantitative differenceswere observed in the earliness of flowering and the percentageof flowering plants. The effects of continuous light and ofshort days with a light break preceding the inductive treatmentwere compared. The results obtained indicate that the inhibitoryeffect of plant age cannot be attributed solely to the appearanceof inhibitors under continuous light but changes of growth patternin plants of different age should also be taken into consideration. The inhibition of RNA synthesis in shoot apices brought aboutby 6-azauridine resulted also in a flowering stimulation, providedthat the inhibitor was applied one or two days prior to inductionand the inductive process itself remained undisturbed. Thisstimulation was accompanied by inhibition of vegetative growthand by a decrease of RNA concentration in the cytoplasm as estimatedcytophotometrically. The competition between growth of vegetative organs and floraldifferentiation affects the response to inductive treatment.The suppression of growth can result in enhancement of flowering.     

Roles of arginine and canavanine in the synthesis and repression of ornithine transcarbamylase by Escherichia coli

Conditions were found under which the processes of repression and derepression of ornithine transcarbamylase were separated from the process of enzyme synthesis. After 10 min of arginine deprivation followed by the addition of 2 to 200 mug of l-arginine per ml, a number of strains of Escherichia coli exhibited a significant burst of ornithine transcarbamylase synthesis which lasted 3 to 4 min before the onset of repression. The rapid increase of enzyme activity was shown to require protein synthesis, and was not due to a slow uptake of arginine or induction of an arginine-inducible ornithine transcarbamylase. The capacity of E. coli to synthesize the burst of ornithine transcarbamylase reached a maximum after 10 min of arginine deprivation and then remained constant. The observed increase in enzyme synthesis may reflect the level of unstable messenger ribonucleic acid (RNA) for ornithine transcarbamylase present in the cell at the time protein synthesis was reinitiated. After the addition of arginine in the absence of protein synthesis, the burst of ornithine transcarbamylase decayed with a half-life of about 3 min. The data implied that arginine prevents synthesis of new messenger RNA that can translate this enzyme. Repression of ornithine transcarbamylase by l-canavanine (100 to 200 mug/ml) was observed, and no active enzyme was formed in the presence of this analogue. The action of canavanine as a repressor was distinguished from the inhibitory effect of this compound on protein synthesis.     

Gibberellins in the control of photoperiodic flower transition in Pharbitis nil

The role of gibberellins in the photoperiodic flower induction of short-day plant Pharbitis nil has been investigated. It has been found that the endogenous content of gibberellins in the cotyledons of P. nil is low before and after a 16-h-long inductive dark period. During the inductive night the content of gibberellins is high at the beginning of darkness and about the middle of the dark period. Exogenous GA₃ when applied to the cotyledons of non-induced plants does not replace the effect of the inductive night but it can stimulate the intensity of flowering in plants cultivated on suboptimal photoperiods. GA₃ could also reverse the inhibitory effect of end-of-day far-red light irradiation on P. nil flowering. 2-Chloroethyltri-methylammonium chloride (CCC) applied to the cotyledons during the inductive night also inhibited flowering. GA₃ could reverse the inhibitory effect of CCC. The obtained results strongly suggest that gibberellins are involved in the phytochrome controlled transition of P. nil to flowering. Their effect could be additive to that of photoperiodic induction.     

Effect of Photoperiod and Chlorogenic Acid on Morphogenesis in Leaf Discs of Streptocarpus Nobilis

Leaf discs from vegetative plants greatly increase their phenolic content when cultivated in vitro. Under long days the values remained constant, and were higher when compared with short days cultures. Under short days total phenolics decreased after 10 d, corresponding to the induction and expression of in vitro flowering. The effect of photoperiod and chlorogenic acid (0.01 mM) on leaf discs cultured from induced and non-induced plants, were analyzed regarding the neo-formation of roots, as well as vegetative and flower buds. Chlorogenic acid enhances the regeneration of roots in all treatments tested, with the highest stimulation on induced leaf discs cultivated in short days. The flowering was not affected by chlorogenic acid, but an inhibitory effect was observed on the neo-formation of vegetative buds in non-induced explants maintained in short days. Vegetative buds were reduced by 50% in flower-induced leaf discs cultivated under short days.     

Nucleic acid synthesis and effect of glucose on its kinetics in cotyledons ofChenopodium rubrum l. during photoperiodic induction

In cotyledons ofChenopodium rubrum L. polydisperse RNA is synthesized in the region of the low molecular weight RNAs during photoperiodic induction. After short-time labelling the rate of 4s RNA synthesis was always higher in induced plants than in plants having obtained a light-break in the middle of the dark period. When glucose was added to the nutrient medium during the dark period of a single photoperiodic cycle the rate of nucleic acid (NA) synthesis was higher in non-induced plants than in induced ones at the termination of the dark period. In plants induced by two cycles in the absence of glucose the rate of NA synthesis at the termination of the second dark period was higher in induced than in non-induced plants. This difference is due to the differential kinetics of NA synthesis during darkness. In plants induced in the presence of glucose the peak of the rhythm in NA synthesis was advanced by 4 h relative to that found in plants induced in the absence of sugar. Thus, the termination of the dark period coincided with the negative slope of the oscillation in plants induced in the presence of glucose, while in plants having obtained a light-break NA synthesis decreased only slightly after having attained its peak. In plants induced in the absence of glucose the termination of the dark period coincided with the peak in the rhythm in NA synthesis. The rhythm in NA synthesis of the cotyledons during the dark period of an inductive cycle is out of phase with the rhythm in flower initiation.     

Changes in Nucleic Acid Metabolism of Excised Barley Leaves During Senescence and the Effect of Kinetin on these Changes

The changes in the amount, rale of synthesis and the nucleotide composition of different RNA fractions in excised barley leaves floated on water or kinetin (10 mg/l) in the dark were examined. In excised leaves floated on water all nucleic acid components declined and these declines were retarded by kinetin. Barley leaves floated on water showed a stimulation of ³²P incorporation into various RNA fractions within 48 hours followed by a decline after 96–144 hours. The leaves floated on kinetin, however, showed an even higher incorporation of ³²P into UNA by 48 hours which remained at a comparatively higher level throughout the experiment. In spite of the above changes in RNA synthesis significant differences in the ³²P sucrose gradient profiles or in the ³²P nucleotide composition of UNA from water and kinetin floated leaves were not noted. The results of this study show that important changes in nucleic acid metabolism occur during the early stages of leaf senescence and that alterations in nucleic acid metabolism during senescence and during kinetin treatment may involve quantitative and only subtle qualitative changes.     

Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

The incorporation of uridine-3H into the shoot apices of photoperiodically induced and non-induced plants ofChenopodium rubrum L.

The influence of photoperiodic induction on the incorporation of uridine-³H into the shoot apices ofChenopodium rubrum was studied using the technique of autoradiography. No increase in uridine incorporation was detected either during induction lasting three days or immediately after its termination. Pyroninophylia likewise did not rise. However, changes in uridine incorporation related to morphogenetic activity during leaf formation and later during differentiation of inflorescences were well marked. The distribution of label in the nucleus immediately after three inductive cycles shows the ratio of extranucleolar to nucleolar incorporation to be higher in non-induced control plants than in induced ones. Data from literature pointing to an activation of RNA synthesis during transition to flowering are discussed and compared with other systems where ontogenetic changes are accompanied by marked changes in RNA synthesis. It is assumed that the activation of RNA synthesis after induction is connected mainly with the activation of growth. However, inChenopodium rubrum photoperiodic induction proceeds together with limited growth and without activation of RNA synthesis.     

Effect of lowered incubation temperatures on nucleic acid and protein synthesis by a mesophilic and a psychrophilic bacterium

The authors studied changes in the synthesis of nucleic acids (RNA, DNA) and protein by a mesophilic strain ofEscherichia coli B and a psychrophilic strain ofPseudomonas fluorescens at a low incubation temperature giving tenfold prolongation of the generation time. It was found that lowering the incubation temperature was followed by an increase in the intracellular nucleic acid content during the lag phase and the phase of accelerated growth, in which maximum nucleic acid (NA) values were reached. As a result, the total NA level in the cell also remained relatively high during further proliferation, when the increase in NA (particularly RNA) slows down at low incubation temperatures. Proteosynthesis, however, fell in the mesophilic culture. The smaller effect of a lowered temperature on DNA biosynthesis was manifested specifically in the lag phase ofEscherichia coli, in which disproportion developed between the amount of DNA (which was synthesized at a relatively higher rate) and RNA; this was afterwards equalized by a temporary break in DNA production. Pronounced differences in the given types of biosynthesis were found only in the mesophilic culture, while at suboptimal temperatures the metabolism of the psychrophilic strain slowed down but no marked changes occurred.