Kinetin and carbohydrate metabolism in chinese cabbage

The effects of kinetin on starch and sugar levels and on ¹⁴CO₂ and ³²P-orthophosphate labeling patterns of floated Chinese cabbage (Brassica pekinensis) leaf discs were investigated. Kinetin caused gross starch degradation. Neutral sugars were depressed by 30 to 40% in leaf tissue treated with kinetin for 24 hours. ¹⁴CO₂ labeling of leaf discs pretreated with kinetin for 24 hours showed increased radioactivity in chloroform-soluble material and most sugar phosphates, and a 35 to 40% decrease in radioactivity in the neutral sugars, glucose, sucrose, and fructose. Incorporation into ATP was increased by 40% by kinetin. ³²P-Orthophosphate uptake was inhibited 30% by kinetin. When corrected for uptake, kinetin stimulated incorporation into chloroform-soluble material but had little effect on other cell fractions. These results indicate that kinetin mobilizes starch reserves and increases the flow of sugars required for the synthesis of lipids and structural materials in floated discs.     

Erratum

Glycolate synthesis was inhibited 40–50% in illuminated tobacco leaf disks, which have rapid rates of photorespiration, when floated on 20 mm potassium glycidate (2,3-epoxypropionate), an epoxide similar in structure to glycolate. The inhibitor also decreased the release of photorespiratory CO₂ about 40%, and the specificity of glycidate was demonstrated by the 40–50% increase in rate of photosynthetic CO₂ uptake observed in its presence. The importance of glycolate synthesis and metabolism in the production of photorespiratory CO₂ and the role of glycolate in diminishing net photosynthesis in species with rapid rates of photorespiration was thus further confirmed. L-(or 2S)-Glycidate was slightly more active than DL-glycidate, but glycidate was more effective as a specific inhibitor in leaf tissue than several other epoxide analogs of glycolate examined. The products of photosynthetic ¹⁴O₂ fixation after 3 or 4 min of uptake were proportionately altered in the presence of glycidate, and the specific radioactivity of the [¹⁴C]glycolate produced was closer to that of the ¹⁴CO₂ supplied. Glycidate inhibited glycolate synthesis in tobacco leaf disks irreversibly, since the degree of inhibition was the same for at least 2 hr after the inhibitor solution was removed. Glycidate also blocked glycolate synthesis in maize leaf disks, tissue with low rates of photorespiration, but large increases in net photosynthesis were not observed in maize with glycidate, because glycolate synthesis is normally only about 10% as rapid in maize as in tobacco. The demonstration of increases in net photosynthesis of 40–50% when glycolate synthesis (and photorespiration) is blocked with glycidate indicates in an independent manner that the biochemical or genetic control of photorespiration should permit large increases in plant productivity in plant species possessing rapid rates of photorespiration.     

Ineffectiveness of cytokinin-induced chlorophyll retention in hypostomatous leaf discs

Kinetin retarded the decrease in chlorophyll content in leafdiscs from 5 species of plants with amphistomatous leaves, wherethe upper surface was exposed to air, but not in Rumex acetosera.When leaf discs were floated so that the lower surface was exposed,the effect of kinetin was less evident. Kinetin also stimulatedtranspiration in leaf discs from Nicotiana tabacum (amphistomatous),but not in leaf discs from Paederia chinensis (hypostomatous).Nor kinetin did retard chlorophyll breakdown in this specieswhen leaf discs were floated so that the stomatal surface wasin contact with the solution. The ineffectiveness of cytokininsin chlorophyll retention in leaf discs from hypostomatous leaveswas not due to reduced uptake of benzylaminopurine-¹⁴C. Chlorophyll retention was severely inhibited by coating theleaf surface with vaseline either in the presence or absenceof kinetin. Leaf discs floated on a solution exposed to CO₂-lessair retained more chlorophyll than those in normal air. Thereis thus a close relationship between stomatal opening (as measuredby stimulation of transpiration) and chlorophyll retention,as influenced by cytokinins. It is suggested that cytokinin-induced chlorophyll retentionand odier effects on leaf tissues could be mediated throughits effects on stomatal opening. (Received January 22, 1976; )     

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.     

Alternate pathways of glycolate synthesis in tobacco and maize leaves in relation to rates of photorespiration

After a preliminary period in light, leaf disks floated on 10 mm α-hydroxy-2-pyridinemethanesulfonic acid to inhibit glycolate oxidase accumulate glycolate at average initial rates of 67 micromoles in tobacco and 8 micromoles per gram fresh weight per hour in maize under optimal conditions in air. In the presence of ¹⁴CO₂, the glycolate synthesized has a high specific radioactivity in illuminated tobacco and a low one in maize. Isonicotinic acid hydrazide also inhibits glycolate oxidation and causes a slow accumulation of glycolate in maize but not in tobacco, while it inhibits glycolate synthesis in tobacco but not in maize. Radioactive carbon in acetate-2-¹⁴C and especially pyruvate-3-¹⁴C is incorporated predominantly into the C-2 of glycolate in both species, but the specific radioactivity is much greater in maize. Glyoxylate-2-¹⁴C is readily converted to glycolate-2-¹⁴C in both species. The addition of phosphoenolpyruvate stimulated glycolate formation in maize and inhibited its synthesis in tobacco, and in the presence of ¹⁴CO₂ the specific radioactivity in glycolate-¹⁴C was decreased greatly by the added phosphoenolpyruvate only in maize.     

Quantification of the kinetin effect on protein synthesis and degradation in senescing wheat leaves

Wheat leaves (Triticum aestivum L. cv San Agustin INTA) were detached when they reached maximum expansion, put individually in tubes containing water and left in darkness. After 3 days the protein content had decreased to 46% of the initial value. When the leaves were placed in 1 micromolar kinetin, they retained 60% of the initial protein content for the same period. This effect was observed only when leaves were treated with kinetin within the first 24 hours after detachment. The action of kinetin on both protein synthesis and degradation was quantitatively measured. Synthesis was estimated by the incorporation of l-[³H]leucine into proteins. It was higher in kinetin treated than in non treated leaves. It contributed to about 14 micrograms of protein retention per leaf in 3 days. Measurement of protein degradation, evaluated by the decay of radioactivity in leaf proteins previously labeled with l-[³H] leucine or as the difference between rates of protein synthesis and protein content, showed that kinetin decreased protein breakdown rates. It accounted for about 186 micrograms of protein retention per leaf in 3 days. Hence, kinetin action on protein breakdown was 13-fold average higher than its action on synthesis for the conservation of leaf protein. This difference is higher in early stages of the process.     

The effect of kinetin on the incorporation of labelled orotate into various fractions of ribonucleic acid of excised radish leaf discs

Summary Discs from senescing radish leaves were floated either on water or on a solution of kinetin and incubated in the dark. The kinetin-treated discs were fed with orotic acid-5-T and the control discs with orotic acid-6-C¹⁴. The two lots of discs were combined and the RNA extracted by the phenol method. The H³:C¹⁴ ratio of the RNA after fractionation on sucrose density gradient was compared with the H³:C¹⁴ ratio of RNA extracted from two lots of control discs, one fed with orotic acid-5-T and the other with orotic acid-6-C¹⁴ and then extracted together. In this way it was found that 21 hours treatment with kinetin causes a small but consistent stimulation of labelled precursor incorporation into all classes of RNA investigated.     

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.     

Studies on Protein Synthesis by Senescing and Kinetin-treated Barley Leaves

Using sterile conditions, changes in total protein synthesis were followed. over an 8 day incubation period, in detached first seedling leaves of barley from 8 day old plants during senescence and after kinetin treatment. In senescing leaves, total ¹⁴C-alanine incorporation was enhanced by nearly 20% within 6 h of leaf detachment and by about 30 % after 24 h. Kinetin treatment stimulated protein synthesis even more, for total incorporation was promoted ca. 50 % after 6 h and by ca. 60 % after 24 h incubation. The leaf supernatant (30,000 ×g for 30 min) proteins were separated on DEAE-Sephadex (A-50) columns into approximately 14 fractions and changes in ¹⁴C labelling of these fractions were studied following leaf detachment and on incubation on water or kinetin for 6 days. In senescing leaves, ¹⁴C-incorporation into supernatant proteins was sustained, even as protein levels declined rapidly The varied stabilities of the different leaf proteins was suggested by the characteristically changing specific activities of the different protein fractions. Although kinetin greatly promoted incorporation into all protein fractions, no evidence was surmised of specific effects on individual leaf proteins. Studies of changes in total protein synthesis in attached senescing first seedling leaves taken from plants aged 7 to 27 days revealed a relatively small increase in ¹⁴C-incorporation. However, incorporation could be greatly increased in leaves up to 15 days old by detaching and preincubating such leaves for up to 2 days on water, prior to measurement. The promotion of ¹⁴C-incorporation into protcins follwing leaf excision could result from early changes in permeability and precursor pool size.     

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.     

Biochemical responses of pea root tissue to cytokinin: enhanced rates of RNA synthesis

Rates of RNA synthesis were studied in cultured pea (Pisum sativum) root segments and cortical explants which require the hormone cytokinin for DNA replication and cell proliferation. Rate calculations were based on the specific radioactivity of the extracted RNA and the specific radioactivity of the extracted ATP pool after a pulse with ³H-adenosine. The kinetics of RNA synthesis was studied after 24 hours of culture with or without kinetin. We found that kinetin stimulated a 2- to 4-fold enhancement in the rate of RNA synthesis after 24 hours of culture as compared to controls. A similar order of magnitude of stimulation of RNA synthesis was found when RNA was isolated by cesium chloride centrifugation. Pulses during the first 24 hours indicate that kinetin stimulates the rate of RNA synthesis as early as 9 hours after treatment has begun. During the first 24 hours of culture, kinetin did not affect the specific radioactivity of the ATP pool. The ATP pool equilibrated slowly with the exogenous label (³H-adenosine) in the presence or absence of kinetin. After 3 days in culture, we found kinetin to cause an expansion of the extractable ATP pool and a corresponding reduction in the ATP pool specific radioactivity. We interpret these results to indicate a stimulation in the rate of RNA synthesis due to kinetin treatment prior to any other known response.     

Melanoprotein Absence of a direct melanin-protein relationship in chick embryp melanocytes

Short-term synthesis of radioactivity labeled melanin (using dl-[2-¹⁴C]tyrosine or 2-[2-¹⁴C]thiouracil) by chick retinal pigment tissues in vitro was not influenced by inhibitors of protein synthesis, puromycin and cyloheximide. Co-ordinate synthesis of protein is, therefore, unnecessary for melanin synthesis, and melanoproteins must represent secondary interactions between melanin and protein. Melanin was isolated from chick embryo feather germs by extracting the proteins with hot dodecyl sulfate/mercaptoethanol. Melanin isolated from tissues incubated previously in l-[U-¹⁴C]valine medium had no associated radioactivity compared to the radioactivity of melanin prepared from tissues incubated in dl-[2-¹⁴C]tyrosine or 2-[2-¹⁴C]thiouracil. If melanoproteins exist at all, they are non-covalently bonded associations of melanin and melanosomal proteins.     

Light-induced h secretion and the relation to senescence of oat leaves

When abraded oat (Avena sativa L. cv Victory) leaf segments are floated on KCl solution, white light causes acidification of the solution external to leaf tissue. The presence of mannitol amplifies the light-induced proton secretion. Mature leaves as well as young ones acidify the medium in light, while senescing leaves (after 3 to 4 days incubated in water in the dark) lose the ability to produce this response to light. The decrease in H⁺ secretion is already measureable after as little as 30 minutes in darkness, while the increase in proteolysis rate was detected only after 6 hours in dark. The decrease in capacity to secrete protons is one of the symptoms of leaf senescence. Moreover, fusicoccin mimics light in stimulating H⁺ pumping and delaying the senescence in the dark. On the other hand vanadate, an apparent inhibitor of plasma membrane H⁺ ATPase, blocks the acidification and promotes the chlorophyll and protein degradation in leaf segments during the 2-day period of incubation. These results, which show a parallel between cessation of H⁺ secretion and acceleration of senescence, may suggest a regulatory role for H⁺ secretion in leaf senescence.     

The effect of Benlate and cytokinins on the content of tobacco mosaic virus in tomato leaf disks and cucumber mosaic virus in cucumber cotyledon disks and seedlings

Tomato leaf disks were inoculated with tobacco mosaic virus (TMV) and floated for 7 days on solutions of kinetin and benzyladenine in the range 20-0-002 mg/1. Virus content was reduced at the higher and increased at the lower concentrations. Benlate and benomyl showed a peak of cytokinin activity in the Amaranthus betacyanin bioassay equivalent to c. 0–002 fig/l kinetin. At concentrations above 25 and 100 mg a.i./l for Benlate and benomyl respectively, both compounds increased the TMV content of tomato leaf disks. Cucumber mosaic virus (CMV) content in cucumber cotyledon disks was increased by Benlate and benomyl treatment (50–100 mg/1). Applied as a soil drench (50–500 mg a.i./l) when the plants were inoculated, Benlate increased the CMV content of infected seedlings. The number of starch-iodide lesions (a measure of susceptibility) was unaltered in cotyledons treated with Benlate 7 days before or immediately after inoculation. Infectivity of crude infective cucumber sap was unaffected by benomyl incorporation, whereas Benlate reduced infectivity at higher concentrations (1000–5000 mg/1). Under the experimental conditions described, Benlate, benomyl, benzyladenine and kinetin had no effect on the chlorophyll content of tomato leaf disks, and intact seedlings.     

Red Light Effects on Uptake of 14C and 32P into Etiolated Corn Leaf Tissue During Photomorphogenic Leaf Opening

Red light effects on the uptake of ¹⁴C and of ³²P was studied by observing leaf sections from 8-day-old etiolated corn leaves that were placed on various substrates following a brief exposure to red radiant energy. There was a general increase of ¹⁴C uptake over dark levels into all metabolite fractions that were prepared. This is in contrast with results obtained previously in which leaf samples were first floated on substrate and then irradiated (Mitrakos et al. 1967, Price et al. 1965). The latter tests resulted in a general decrease in sugar and starch as well as ¹⁴C content of all fractions. However, under both types of experimental conditions the red light effect manifested itself as an increase in hexosemonophosphate turn-over rate and accumulation of radioactivity in the cell wall polysaccharide fraction. The present data further substantiate the previous work in that they demonstrate the regulatory influence of the hexose pool size on the intermediary metabolism and the manifestation of the phytochrome responses. From the data thus far obtained it cannot be determined as to whether or not phytochrome mediates by controlling phosphorylating activity or through control of specific enzymatic processes that lead to observable cell wall polysaccharide synthesis and leaf unrolling.     

Kinetin Reversal of NaCl Effects

Leaf discs of Nicotiana rustica L. were floated on NaCl in the presence of kinetin or abscisic acid. On the 5th day ¹⁴CO₂ fixation, [³H]leucine incorporation, stomatal conductance, and chlorophyll content were determined. Kinetin either partially or completely reversed the inhibitory effects of NaCl while ABA had no effect.     

Inhibition of the Development of a Cation Accuniulatory System and of Tris-induced Uptake in Storage Tissues by N6-benzyladenine and Kinetin

The effect of N⁶-benzyladenine (BA) and kinetin on cation uptake in disks of beetroot (Beta vulgaris L.) and swede (Brassica napobrassica Mill.) tissue was measured in aerated solutions, containing 1 mM NaCl or 1 mM KCl, and in the presence or absence of trishydroxymethyl ammo methane (tris) buffer at pH 8. This investigation followed a suggestion that the immediate activation of cation uptake in freshly sliced beetroot disks by tris (the tris-effect) may depend on stimulation of hexokinase activity. BA, a competitive inhibitor of hexokinase, caused a complete inhibition of tris stimulated cation uptake in beetroot disks, but the effect was delayed and preceded by a 3–4 hour period of promoted cation uptake. Generally, the effects of BA and kinetin were identical. Use of ¹⁴C-kinetin indicated a rapid incorporation of kinetin into the tissue. BA and kinetin also prevented the development of a cation uptake capability during the aging of the beetroot disks. Swede disks which do not show a tris-effcct and no lag phase in development of cation uptake capability were not affected by BA and kinetin treatments. Mechanisms of tris induced cation uptake are discussed with emphasis on its role as a proton acceptor.     

Pyridine salvage and nicotinic acid conjugate synthesis in leaves of mangrove species

The metabolic fate of [carbonyl-¹⁴C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [¹⁴C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-¹⁴C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3 h after administration. Radioactivity from [carbonyl-¹⁴C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.     

Comparison of adenosine metabolism in leaves of several mangrove plants and a poplar species

A possible increased demand for ATP in salt- tolerant mangrove plants was studied by the comparison of metabolic fates of [8-¹⁴C] adenosine in leaf disks of several mangrove plants and of poplar. In mangrove trees, Rhizophora stylosa, Bruguiera gymnorrhiza, Kandelia candel and Sonneratia alba, 56–92% of [8-¹⁴C]adenosine taken up by leaf disks was converted during 3 h incubation to salvage products, i.e., nucleotides and RNA. Synthesis of nucleotides including ATP was stimulated by salt stress induced by 250 mM NaCl. In leaf disks of Avicennia marina, a mangrove shrub that produces glycinebetaine as compatible solutes, 46% of radioactivity entered salvage products when [8-¹⁴C] adenosine was continuously supplied to the leaf disks. Hydrolysis of adenosine to adenine was extremely active in this mangrove shrub. This is probably due to the high activity of adenosine nucleosidase (EC 3.2.2.7). In leaf disks of another mangrove shrub, Lumnitzera racemosa, only limited amounts of [8-¹⁴C]adenosine were metabolised (< ca. 30% taken up by leaf disks), but synthesis of ATP and ADP was stimulated by salt stress. In Pemphis acidula leaf disks, adenosine salvage activity was low and more than 30% of adenosine was hydrolysed to adenine. In leaf disks of poplar, a non-salt-resistant plant, ca. 40% of [8-¹⁴C] adenosine was converted to salvage products during 3 h of incubation, but the rate was slightly reduced by treatment with 250 mM NaCl. The present results suggest that large mangrove trees generally have efficient adenosine salvage ability, which is stimulated by salt. Lesser salvage activity is found in small size mangrove shrubs, although salt generally still enhances salvage activity.     

The hormonal control of betacyanin synthesis in Amaranthus caudatus

Gibberellic acid (GA₃) inhibits amaranthin synthesis whereas the growth retardant, phosphon D, enhances pigment levels in A. caudatus seedlings exposed to light. No effect was observed on chlorophyll and carotenoid synthesis. Radioactive tyrosine and DOPA were incorporated into amaranthin. The specific activity of amaranthin synthesised in the presence of ¹⁴C-tyrosine or ¹⁴C-DOPA in seedlings treated with GA₃ is higher than water controls. The specific activity of pigment from phosphon D treated tissue is relatively low. GA₃ treated tissue has lower active tyrosine and DOPA pools compared to phosphon treated seedlings. Tyrosine and DOPA-oxidase activity increases in GA₃ treated and H₂O control seedlings exposed to light. Kinetin stimulates the synthesis of amaranthin in dark-grown seedlings and this is not overcome by simultaneous GA₃ application. Dark-grown seedlings treated with different kinetin concentrations and incubated in ¹⁴C-tyrosine synthesise radioactive amaranthin of similar specific activity. Kinetin treatment of dark-grown seedlings brings about an increased tyrosine and DOPA-oxidase activity. The results indicate that GA₃ controls the production and/or availability of tyrosine whereas kinetin can mimic light treatment and controls the utilisation of tyrosine probably by bringing about the synthesis or activation of tyrosine and DOPA-oxidase protein.