AUXIN-INDUCED GROWTH OF TUBER TISSUE OF JERUSALEM ARTICHOKE: II. THE RELATION TO PROTEIN AND NUCLEIC ACID METABOLISM

The following results were obtained using tissue slices excisedfrom cold-stored Jerusalem artichoke tuber. 1. Increase in protein content of the tissue was small duringthe washing (i.e. "aging"), and great in the growth phase, particularlyin washed tissue. 2. RNA content of tissue increased during the growth periodsimilarly in non-growing tissue (in water) and actively growingtissue (in 2,4-D plus KIN). 3. Both RNA and DNA increased during the washing, the increasebeing greater in RNA than in DNA. This RNA increase was enhancedby gibberellic acid. 4. 2-Thiouracil, 8-azaguanine, puromycin, and mitomycin C givenat the washing inhibited the subsequent growth. The effect ofthese inhibitors was not significant when they were given inthe growth period. 5. Mitomycin C reduced the basophilia of nuclei and made themswell, as did deoxyribonuclease. 6. The effect of inhibitors of nucleic acid metabolism was reversedto some extent by gibberellic acid and by kinetin. 7. Chloramphenicol inhibited the growth strongly if given inthe growing period, but not so strongly if given during thewashing. 8. An autoradiographic study using ³H-cytidine suggested thatRNA is synthesized in nucleus during the period of washing andis transferred to cytoplasm via nucleolus. It is conjectured that the RNA synthesized during the agingis responsible for the expansion growth to be caused later byauxin or auxin plus kinetin. (Received September 4, 1965; )     

AUXIN-INDUCED GROWTH OP TUBER TISSUE OP JERUSALEM ARTICHOKE: III. EFFECT OF ACTINOMYCIN D ON AUXIN-INDUCED EXPANSION GROWTH AND RNA SYNTHESIS

1. The following results were obtained using tissue slices excisedfrom cold-stored Jerusalem artichoke tubers.
2. Actinomycin Dat the concentration of 20 µg/ml given duringthe agingperiod did not affect the subsequent expansion growthcausedby auxin or auxin plus kinetin.
3. Actinomycin D given in thegrowth period, on the other hand,strongly inhibited the expansiongrowth of tissue slices agedin the absence of the antibiotic.
4. In the growth period, auxin or auxin plus kinetin promotedtheincorporation of uracil-2-¹⁴C into RNA fraction.
5. ActinomycinD inhibited the incorporation of ³²P orthophosphateinto ribosomalRNA during the aging period.
6. In the growth period, the incorporationof ³²P into RNA wasenhanced by auxin and was inhibited by actinomycinD, more remarkablyin ribosomal RNA than in lighter RNA.

¹A part of this paper was presented at the Conference on PlantGrowth Regulators held by the New York Academy of Sciences onMay 16, 1966.     

Changes in enzyme activity during elongation and tuberization of stolons of Solanum tuberosum L. cultured in vitro

Isolated stolons of Solanum tuberosam L. were cultured in vitroin the presence of kinetin, which induced tuber initiation orgibberellic acid which inhibited initiation. Progressive changes in enzyme activity, at the locus of tuberinitiation, were monitored at specified intervals. In the presenceof kinetin soluble invertase activity was decreased with timewhereas gibberellic acid (GA) evoked substantial increases inactivity. Acid phosphatase activity was enhanced by GA but changedonly slightly under tuber inducing conditions. Enzymic hydrolysisof glucose-6-phosphate and fructose-6-phosphate decreased duringthe course of tuber induction but increased in the presenceof GA. In contrast hydrolysis of 3'AMP was stimulated undertuber inducing conditions. GA evoked substantial increases in peroxidase activity duringthe initial stages of incubation while under tuber inducingconditions increased activity was only observed after 5 days.Substantially higher levels of IAA oxidase activity were associatedwith tuber initiation. RNase activity decreased with time undertuber inducing conditions but showed an initial stimulationin the presence of GA. These results are discussed with referenceto the role of these enzymes in carbohydrate metabolism andthe regulation of hormone levels. (Received February 29, 1972; )     

Effect of Kinetin on Ribosomes of Excised Barley Leaves

The changes in the amount and the composition of ribosomes in excised barley leaves floated on water or on 10 mg/l kinetin solution in the dark were examined. The rapid loss of polyribosomes and ribosomes in leaves floated on water was greatly retarded by kinetin. The ribosomes-polyribosomes which originally contained 49 per cent protein showed substantial decline in protein content in leaves floated on water but only slight decline in leaves floated on kinetin solution. It is suggested that kinetin by stimulating RNA synthesis and by suppressing the activities of rihonuclease and peplidase may preserve the ribosomes in excised leaves.     

The Initiation, Growth, and Characteristics of a Tissue 2

The rate and extent of initiation of callus from potato tuberdiscs depends on the concentrations of auxin and kinetin inthe medium on which they are grown. NAA is the most effectiveauxin, initiating callus at a concentration (0. 01 mg/1) anorder of magnitude lower than for IAA or 2,4-D. There is a week'slag before initiation begins with IAA or 2,4-D. In combinationwith each auxin, kinetin is inhibitory to initiation of callusand its growth on the explant. High-intensity light and lowtemperature are also inhibitory. In isolated callus subcultured so as to prevent dilution ofits accumulated auxin, the only effect of varying exogenousauxin levels is as a progressive inhibition by NAA. If thisdilution is permitted, however, 2,4-D and IAA have an optimumgrowth promoting activity at 1 mg/1, whereas the effect of NAAincreases up to 10 mg/1. The growth of the callus is affectedby agar concentration (1 per cent optimum), and is halted bypH values below 5. The callus grows on various carbon sourcesbut is dependent upon one or more components of N. Z. Amine;it also requires a number of micronutrients. A suspension culture from the callus exhibits the usual growthcurve. The phenolic content follows a pattern different fromthat of growth, protein, and RNA content, and phenolics arerapidly synthesized as active growth ceases. In contrast tothe callus tissue, the suspension culture grows at a wide rangeof pH values and buffers the medium. At low temperatures in the light, potato discs produce greencallus with a chlorophyll content corresponding to that of thediscs from which they grew. The isolated callus tissue doesnot require kinetin and produces and excretes its own cytokinin(s).The amount synthesized varies over the growth cycle.     

Studies on the Physiology of Rice: XV. CHANGES IN THE NITROGEN METABOLISM OF SEEDS DURING GERMINATION AND THEIR RELATION TO THE APPLICATION OF GROWTH REGULATORS

Seeds of rice variety ‘Chinsurah Boro’ were treatedwith two concentrations of indoleacetic acid (IAA) and maleichydrazide (MH), 0.1 mg. per litre and 0.01 mg. per litre, duringthe first 7 days of germination, and the resulting changes innitrogen metabolism of both endosperm and embryo were determined.The seedling growth at different times during the treatmentwas recorded. Uptake of water by the embryo increased up to 72 hours, butthereafter the percentage water content fell, owing to large-scaletranslocation of dry matter to it. The endosperm showed increasingwater content for 5 days. There are two marked stages of nitrogen metabolism of the seedduring germination. During the first 72 hours hydrolysis ofprotein in the endosperm and translocation of soluble nitrogento the embryo are the predominant features. Thereafter the embryoactively synthesizes protein from the products of translocation. IAA and MH affected the seedling growth similarly. Initiallythere was a small retardation of leaf growth, but at later periodsrecovery took place. Root growth was more adversely affectedthan shoot growth, the effect also persisting longer. Thesechanges in growth were reflected in the nitrogen metabolismof the seedlings. For the first few days IAA and MH retardedthe supply of soluble nitrogen from the endosperm to the embryo,consequently there was less soluble nitrogen in the embryo thoughprotein synthesis there was affected only slightly. The differencein soluble nitrogen between the treated and untreated embryospersisted throughout the seven days. An attempt is made to explainthis on the basis that primarily IAA and MH retard the enzymaticactivity responsible for the hydrolysis of protein in the endosperm.     

Nucleic acid and protein synthesis in discs cut from mature leaves of Nicotiana tabacum L. and cultured on nutrient agar with and without kinetin

The effect of kinetin on aspects of the metabolism of discs cut from mature leaves of Nicotiana tabacum and cultured in the light on agar containing mineral salts and sucrose was studied. In the first few days of culture there was a rapid decline in chlorophyll content. Discs treated with kinetin in the light began to resynthesise chlorophyll after 3–4 days and this was correlated with chloroplast replication. Kinetin promoted chloroplast replication but was not always essential. An increase in fresh weight also occurred, due mainly to cell expansion. Nitrate reductase activity increased rapidly during the first few hours after placing discs on the culture medium but kinetin had no effect on this reponse. Subsequently there were dramatic increases in RNA and protein content which were largely independent of kinetin. Gel electrophoresis showed that cytoplasmic and chloroplast ribosomal RNA and a large amount of soluble RNA were synthesised during culture of the discs. These results are discussed in relation to the role of kinetin in delaying leaf sensescence.     

Effects of sucrose and kinetin on growth and chlorophyll synthesis in tobacco tissue cultures

Investigations were carried out on the effects of various combinations of sucrose and kinetin concentrations on growth and chlorophyll production in a green and a nongreen clone of pith callus of Nicotiana tabacum L. It was found that 2 milligrams per liter or higher amounts of kinetin induced greening in the nongreen tissue. The observations suggested that growth of the callus and synthesis of chlorophyll and soluble protein are controlled by relative concentrations of sucrose and kinetin in the medium. Kinetin was found to be inhibitory for chlorophyll synthesis in the green callus.     

Studies on the mechanisms of ozone tolerance: Cytokinin-like activity of N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea, a compound protecting against ozone injury

The cytokinin-like activity of the growth regulating chemical EDU, N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea, was determined and compared with the actitivity of kinetin using the tobacco callus bioassay. EDU has a pronounced stimulatory effect on callus growth at concentrations of 5 × 10⁻⁴ and 1 × 10⁻³ M but was 5 000 times less potent than the synthetic cytokinin, kinetin. Senescence regulation and oxidant resistance induced by EDU and kinetin were also studied. EDU retarded the breakdown of chlorophyll, protein and RNA in 0₃-sensitive tobacco leaf discs during senescence. EDU was much more effective in arresting senescence and in protecting against 0₃ injury than kinetin. Results indicate the EDU-induced plant tolerance to 0₃ phytotoxicity may be indirect through enzyme induction regulation.     

Über die Wirkung kurzfristiger Kinetingaben auf Protein,Aminosäuren und RNS bei Lemna minor L.

Summary Lemna minor L. was cultivated on nutrient medium with kinetin (10^-5 m) for 3 hours at 28°C and ca. 5,000 lux. Samples were taken at different times and their amino acid, protein and RNA content was determined.The amino acid content in the cultures with kinetin corresponds to that in the cultures without kinetin. The amino acid concentration seems to be regulated by mechanisms independent of kinetin. On the other hand, kinetin increases the quantity of protein immediately i.e., within the first two minutes. This increase stops after ca. 30 minutes, but the excess of protein remains for a long time. In the first 30 minutes the RNA concentration also rises. This increase is not as strong as that of the protein nor does it remain. After 30 minutes, when the rise of protein content stops, the RNA concentration begins to drops within 1 hour to that of the control cultures without kinetin.The extent of the protein and RNA increase induced by kinetin depends strongly upon the physiological state of the plants. This may be because the plants produce different quantities of endogenous cytokinins under different conditions.The protein induced by kinetin was then examined after centrifugation at 100,000 x g. In samples taken during the first 5 minutes, the quantity of protein in the supernatant and in the sediment is increased. In samples taken after this period, the increase of protein in the supernatant disappears and only that in the sediment remains. After some time, therefore, all the excess protein consists of structural protein, which is found in the sediment. The findings are compared with those of other authors and an unspecific stimulation of RNA is suggested.     

The effects of abscisic acid,kinetin and 5-fluorouracil on ribonucleic acid and protein synthesis in senescing radish leaf disks

Summary The effects of abscisic acid and kinetin on RNA synthesis in senescing radish leaf disks were investigated using the improved resolution afforded by polyacrylamide gel electrophoresis. Kinetin stimulated and abscisic acid inhibited incorporation of radioactivity into cytoplasmic ribosomal RNA and soluble RNA. Chloroplast ribosomal RNA synthesis appeared to be confined to the period of leaf expansion and was not detected in fully mature leaves. The effects of kinetin in retarding and of abscisic acid in accelerating leaf senescence were not altered by the inhibition of cytoplasmic ribosomal RNA synthesis with 5-fluorouracil. Following inhibition of cytoplasmic ribosomal RNA synthesis with 5-fluorouracil, kinetin stimulated and abscisic acid inhibited incorporation of radioactivity into polydisperse RNA. These results are discussed in relation to the possible mode of action of kinetin and abscisic acid in senescing leaf tissue.     

The Changes in Soluble Protein of Excised Barley Leaves during Senescence and Kinetin Treatment

Sterile detached barley leaves were floated on water or kinetin (10 mg/1)and supernatant extracts (30,000 x g for 30 min) were prepared from these leaves over an 8 day incubation period. Changes in selected total enzyme levels and in individual soluble protein components wore compared. Ribonuclease, deoxyribonuclease and peptidase activities rose in senescing leaves, even as total protein levels fell. Kinetin to some extent depressed these activities. Evidence of considerable loss of ribonuclease and to a lesser extent of deoxyribonuclease into the surrounding medium was obtained. Soluble supernatant ant extracts were resolved on DEAE-Sephadex (A-50) columns into about 15 components. While most components were degraded during senescence they did so at different rates. Kinetin lowered the rate of degradation of all components. Since no conclusive evidence of a new protein(s) was obtained in water and kinetin treated leaves, it was considered that any protein changes may have been of a quantitative rather than qualitative nature.     

Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

Kennzeichnung der Bildung und Alterung von Wurzeln in Callus-und Organ-Kulturen von Daucus carota durch die Aktivität der Glutamatdehydrogenase

Summary In callus tissues of Daucus carota rhizogenesis was influenced by addition to the cultures of solutions with different kinetin concentrations (0–0.1–0.5–2 mg/l) and a constant auxin content (1 mg/l). In these cultures the specific activity of glutamate-dehydrogenase (E.C. 1.4.1.2.), aspartate-aminotransferase (E.C. 2.6.1.1.), isocitrate-dehydrogenase (E.C. 1.1.1.42) and of acid phosphatase (E.C. 3.1.3.2.) was determined. Before root initiation can be seen morphologically in tissues grown on low kinetin concentrations, the specific activity of glutamate-dehydrogenase increases to more than three times the level found in cultures with no subsequent root initiation, whereas the specific activity of the other three enzymes changes far less. Total soluble protein measured as percentage of fresh weight remains nearly the same in all callus cultures. The activity of the same enzymes was measured in liquid grown roots of Daucus carota during a period of ageing of 130 days. During this time, the specific activity of glutamate-dehydrogenase is reduced to 1/10 of the value found in growing roots, whereas the activity of the other three enzymes is reduced only to 1/2 or to 1/3 of this value. Therefore, the state of senescence and the capacity for further growth can be characterized by the specific activity of the glutamate-dehydrogenase in the roots. The changes in the specific activity of glutamate-dehydrogenase in callus tissue and in the roots do not depend on inhibitory substances in the cell-free extracts.     

Characterization of Sertoli cell RNA synthetic activities in vitro at selected times during sexual maturation

In this study, Sertoli cell RNA synthetic activity in vitro was characterized at selected times during sexual maturation. Sertoli cells, isolated from rat testes undergoing the first wave of spermatogenesis and placed in culture for 4 days, exhibited 2-fold increases in soluble ribonucleotide pools and in total RNA concentrations over the age span of 18-35 days. High performance liquid chromatographic analysis of the ribonucleotide pools in Sertoli cells cultured from 18- and 33- to 34-day-old rats revealed that, in addition to the overall age-related doubling of concentrations, uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools were disproportionately increased 4- and 6-fold, respectively. In general, Sertoli cell contained relatively small amounts of UTP in comparison to several other cell types, but exhibited a high ADP:ATP ratio. A uniform 2-fold increase in the base composition of Sertoli cell RNA per mg DNA was observed over the age span of 18-35 days, with no preferential increase in any one specific nucleotide. There was no change in [3H]uridine incorporation (2 h) into RNA per cell (pmol/mg DNA), but decreased specific activity of the RNA (pmol/mg RNA) in Sertoli cells cultured from 35-day-old rats as compared to those from 18- to 19-day-old rats. Similar differences were noted in the specific activity of label incorporated into specific RNA bases. In contrast, the specific activity of the UTP-CTP soluble pool/mg DNA was only slightly increased. These data indicate that processes related to RNA synthesis in the Sertoli cell undergo a number of changes during the period of sexual maturation.     

Ribulose-1,5-Diphosphate Carboxylase Protein during Flag Leaf Senescence

Ribulose-l,5-diphosphate (RuDP) carboxylase protein and activitywere determined in relation to net photosynthetic rate duringthe senescence of intact flag leaves of wheat on the plant.Initially the decrease in RuDP carboxylase activity was greaterthan the decline in net photosynthesis. The major decrease inRuDP carboxylase activity over this period resulted from a decreasein enzyme specific activity from 11 to 2 µmol CO₂ fixedh^–1 mg^–1 protein. Loss of RuDP carboxylase proteindid not occur until late in senescence by which time chlorophyllconcentration had decreased by more than 50%. Treatment of flagleaves at weekly intervals with either 1000 parts 10^–62-chloro-ethyltrimethylammonium chloride or 100 parts 10^–6gibberellic acid with 1 part 10^–6 kinetin did not significantlyaffect net photosynthetic rate, RuDP carboxylase protein oractivity during senescence.     

Effects of Cytokinins on the Nucleic Acids of Tobacco Pith

Cylinders of pith of Nicotiana tabacum var. Wis. 38 were asepticallyisolated and grown on a mineral-sucrose medium in the presenceand absence of either kinetin or zeatin. On medium containing kinetin (5x10⁶M) the increase in the residualdry weight of cylinders was greater than that on control mediumafter 2 d culture and this was maintained at 5 d. At 2, 5, and7 d there were increments in the DNA and RNA levels of cylindersdue to kinetin treatment and these increased progressively. A double-labelling method coupled with acrylamide gel electrophoresiswas used to study the effects of kinetin at 5 x 10^–6Mand zeatin at 10^–6 M on the incorporation of radioactiveprecursors into the RNA of cylinders given a 4-h label pulse.No effects of zeatin were observed after 1 d nor of kinetinat 2 d but after 4 d zeatin stimulated incorporation into ribosomal,transfer and poly-disperse RNA and kinetin did likewise after5 d. Incorporation measured at 7 d was reduced in cylindersfrom which kinetin had been removed at 5 d compared to thoseto which it had been supplied continuously. Scans at 260 run of the electrophoretic fractionations of RNAafter 2, 5, 7, and 9 d of culture showed that both in the absenceand in the presence of kinetin the proportion of 4S RNA increasedfrom about 20 per cent to over 50 per cent of the total between2 and 5 d of culture and after 5 d the ribosomal RNA gave aberrantscans apparently indicative of degradation. These aberrant scanspersisted in RNA extracted after 27 d culture in both the presenceand absence of kinetin.     

On the interaction of growth retardants with IAA and kinetin

In the pea test a highly positive response to the treatment with IAA reversed to a negative one or became 5 to 6 times weaker when CCC was applied together with IAA. In cultivating pea seedlings, following their decapitation, for two days in a 0.25 per cent CCC solution and then in water, growth of their cotyledonous axillaries (cotylaries) were inhibited. This inhibitive action of CCC could be made ineffective when the seedlings, following two-days’ cultivation in the CCC solution, were grown further in kinetin solutions (0.37–3 mg per 1). Cotylaries of decapitated pea seedlings, when grown in kinetin solutions were inhibited. With kinetin solutions of 6–12 mg/l a strong inhibition also occured in the growth of roots at the apical parts of which spherical swellings were developing. The CCC supplied to the roots of intact etiolated pea seedlings is translocated acropetally into the stem at a rate of about 5 cm per hour. Decapitation of the plant causes retardation of this transport, yet a coat of 0.00001–1% IAA or kinetin paste produces acceleration of the stream. Existence of an antagonism between CCC and IAA, demonstrated earlier, was found holding true also for B-9 (N, N-dimethyl-aminesuccinamic acid) and IAA, as the inhibitive action of B-9, 0.06% solution on the growth of lettuce hypocotyls was reduced to a highly significant degree when the plants were supplied with B-9 together with IAA at a concentration of 10 mg/l.     

An improved method for micropropagation and regeneration of date palm (Phoenix dactylifera L.)

We developed a novel large-scale micropropagation pathway for date palm (Phoenix dactylifera L.) based on organogenesis. We obtained organogenic stems from shoot tip explants of the Moroccan date palm cultivar Najda, and investigated shoot proliferation from these organogenic stems in vitro on various media; Beauchesne medium (BM) and Murashige and Skoog medium (MS) at full-strength, half-strength, and one-third-strength, containing various concentrations (0, 0.25, 0.5, and 1 mg/L) of 2-naphthoxyacetic acid (NOAA) and kinetin. The optimal medium during the multiplication phase was half-strength Murashige and Skoog medium (MS/2) supplemented with 0.5 mg/L NOAA and 0.5 mg/L kinetin (23.5 morphologically superior shoots per explant, with low vitrification rates). For the shoot elongation phase, shoots were transferred to the same proliferation medium, or to MS or MS/2 media without plant growth regulators (PGRs). Shoots elongated rapidly and showed a high rate of root formation on media supplemented with PGRs. For example, on MS/2 medium containing 1 mg/L NOAA and 1 mg/L kinetin, the average shoot length was 15.1 cm, the average number of roots per shoot was 6.2, and their average length was 3.4 cm. On PGR-free media, shoots were shorter with wider and greener leaves, and had fewer roots. The plantlets were transferred to a greenhouse for acclimation. The survival rate after 2 months was related to the medium used during the elongation phase; >90 % of shoots that were cultured on PGR-free media survived, while there was a poor survival rate of shoots that had been cultured on media containing PGRs.     

Isoperoxidases in jerusalem artichoke in relation to tuberization and dormancy

Peroxidase activity and isoenzyme pattern were investigated in buds and tubers of Jerusalem artichokes in relation to induction and breaking of dormancy. Peroxidase activity per unit soluble protein is the highest in the dormant stage. Conditions leading to growth,i.e. release of dormancy by the cold, stimulation of axial growth by gibberellic acid or stimulation of radial growth (tuberization) by kinetin, cause rapid loss of total peroxidase activity together with a decrease of intensity of the most cathodic isoperoxidases. Induction of dormancy by AMO-1618 increases peroxidase activity mainly through the same cathodic isoenzymes. The role of the cathodic isoperoxidases is discussed in relation to auxin catabolism and the genesis of oxygenation products inhibitory to plant growth.