DNA SYNTHESIS,CELL DIVISION,AND CELL DIFFERENTIATION DURING LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

Leaf growth consists of two basic processes, cell division and cell enlargement. DNA synthesis is an integral part of cell division and can be studied with autoradiographic techniques and incorporation of some labeled precursor. Studies were made on the synthesis of nuclear DNA through incorporation of ³H-thymidine in various parts of the lamina during the entire course of leaf development of Xanthium pennsylvanicum. The time course analysis of DNA synthesis was correlated with cell division and rates of cell enlargement. Significant differences in ³H-thymidine incorporation were found in various parts of the lamina. Cell division and DNA synthesis were highest in the early stages of development. Since no ³H-thymidine was incorporated after cessation of cell division (LPI 2.8) in the leaf lamina, it appears that DNA synthesis is not needed for enlargement and differentiation of Xanthium cells. Rates of cell enlargement were negligible in the early development and reached their maximum after cessation of mitoses, between plastochron ages (LPI) 3 and 4. Cells matured between LPI's 5 and 6. Enzymatic activity was correlated with cell division and cell differentiation at various stages of leaf development.     

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

Structure and Biosynthesis of Cuticular Lipids: Hydroxylation of Palmitic Acid and Decarboxylation of C(28), C(30), and C(32) Acids in Vicia faba Flowers

The structure and composition of the cutin monomers from the flower petals of Vicia faba were determined by hydrogenolysis (LiAlH₄) or deuterolysis (LiAlD₄) followed by thin layer chromatography and combined gas-liquid chromatography and mass spectrometry. The major components were 10, 16-dihydroxyhexadecanoic acid (79.8%), 9, 16-dihydroxyhexadecanoic acid (4.2%), 16-hydroxyhexadecanoic acid (4.2%), 18-hydroxyoctadecanoic acid (1.6%), and hexadecanoic acid (2.4%). These results show that flower petal cutin is very similar to leaf cutin of V. faba. Developing petals readily incorporated exogenous [1-¹⁴C]palmitic acid into cutin. Direct conversion of the exogeneous acid into 16-hydroxyhexadecanoic acid, 10, 16-dihydroxy-, and 9, 16-dihydroxyhexadecanoic acid was demonstrated by radio gas-liquid chromatography of their chemical degradation products. About 1% of the exogenous [1-¹⁴C]palmitic acid was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes, which were identified by combined gas-liquid chromatography and mass spectrometry as the major components of the hydrocarbons of V. faba flowers. The radioactivity distribution among these three alkanes (C₂₇, 15%; C₂₉, 48%; C₃₁, 38%) was similar to the per cent composition of the alkanes (C₂₇, 12%; C₂₉, 43%; C₃₁, 44%). [1-¹⁴C]Stearic acid was also incorporated into C₂₇, C₂₉, and C₃₁n-alkanes in good yield (3%). Trichloroacetate, which has been postulated to be an inhibitor of fatty acid elongation, inhibited the conversion of [1-¹⁴C]stearic acid to alkanes, and the inhibition was greatest for the longer alkanes. Developing flower petals also incorporated exogenous C₂₈, C₃₀, and C₃₂ acids into alkanes in 0.5% to 5% yields. [G-³H]n-octacosanoic acid (C₂₈) was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes. [G-³H]n-triacontanoic acid (C₃₀) was incorporated mainly into C₂₉ and C₃₁ alkanes, whereas [9, 10, 11-³H]n-dotriacontanoic acid (C₃₂) was converted mainly to C₃₁ alkane. Trichloroacetate inhibited the conversion of the exogenous acids into alkanes with carbon chains longer than the exogenous acid, and at the same time increased the amount of the direct decarboxylation product formed. These results clearly demonstrate direct decarboxylation as well as elongation and decarboxylation of exogenous fatty acids, and thus constitute the most direct evidence thus far obtained for an elongation-decarboxylation mechanism for the biosynthesis of alkanes.     

Biosynthesis of pentahydroxystearic acid of cutin from linoleic acid in Rosmarinus officinalis

The hydroxyfatty acid polymer, cutin, is the structural component of plant cuticle. Combined gas chromatography-mass spectrometry of the hydrogenolysis and deuterolysis products of rosemary cutin (Rosmarinus officinalis) revealed a series of components suggesting the conversion of linoleic acid to 9,10,12,13,18-pentahydroxy-stearic acid. [U-¹⁴C]Linoleic acid was incorporated into the insoluble residue of rapidly expanding rosemary leaves. Depolymerization of the insoluble material followed by isolation of individual components and chemical degradation studies showed that linoleic acid was directly converted into 18-hydroxylinoleic acid, 18-hydroxy-9, 10-epoxyoctadec-12-enoic acid, 9,10,18-trihydroxyoctadec-12-enoic acid, 9,10,18-trihydroxy-12,13-epoxystearic acid, and 9,10,12,13,18-pentahydroxystearic acid. These results strongly suggest that, in the biosynthesis of the phytopolymer, linoleic acid is first converted into 18-hydroxylinoleic acid and that this precursor then undergoes sequential epoxidation-hydration at the Δ⁹ and Δ¹² double bonds to yield 9,10,12,13,18-pentahydroxystearic acid.     

Cold-induced fructosan synthesis in leaves of Dactylis glomerata

Dactylis glomerata accumulated fructosan more rapidly at 5° than at 15—20°. The pattern of incorporation of ¹⁴CO₂ into fructosan was determined in plants grown at 5°. During the major period of fructosan synthesis there was initial incorporation of label into mono- and disaccharides, and progressive synthesis of polymeric material occurred subsequently. Rates and levels of synthesis were much lower in leaf blades than in leaf bases. The MW distribution of the polymeric material in leaf bases differed from that in the blades and from that observed in plants which synthesize inulin.     

A functional cutin matrix is required for plant protection against water loss

The plant cuticle, a cutin matrix embedded with and covered by wax, seals the aerial organ''s surface to protect the plant against uncontrolled water loss. The cutin matrix is essential for the cuticle to function as a barrier to water loss. Recently, we identified from wild barley a drought supersensitive mutant, eibi1, which is caused by a defective cutin matrix as the result of the loss of function of HvABCG31, an ABCG full transporter. Here, we report that eibi1 epidermal cells contain lipid-like droplets, which are supposed to consist of cutin monomers that have not been transported out of the cells. The eibi1 cuticle is fragile due to a defective cutin matrix. The rice ortholog of the EIBI1 gene has a similar pattern of expression, young shoot but not flag leaf blade, as the barley gene. The model of the function of Eibi1 is discussed. The HvABCG31 full transporter functions in the export of cutin components and contributed to land plant colonization, hence also to terrestrial life evolution.Key words: ABC transporter, cuticle, cuticular wax, drought resistance, inclusion     

Topograph,a Software Platform for Precursor Enrichment Corrected Global Protein Turnover Measurements

Defects in protein turnover have been implicated in a broad range of diseases, but current proteomics methods of measuring protein turnover are limited by the software tools available. Conventional methods require indirect approaches to differentiate newly synthesized protein when synthesized from partially labeled precursor pools. To address this, we have developed Topograph, a software platform which calculates the fraction of peptides that are from newly synthesized proteins and their turnover rates. A unique feature of Topograph is the ability to calculate amino acid precursor pool enrichment levels which allows for accurate calculations when the precursor pool is not fully labeled, and the approach used by Topograph is applicable regardless of the stable isotope label used. We validate the Topograph algorithms using data acquired from a mouse labeling experiment and demonstrate the influence that precursor pool corrections can have on protein turnover measurements.Methods of measuring protein synthesis and degradation using stable or radioactive isotope labels have existed for decades. The isotope label is introduced in the form of a labeled amino acid or amino acid precursor, and the incorporation or removal of that label from protein is used to estimate average protein turnover rates (1, 2). Historically, the amount of stable isotope label incorporated into a protein is measured by enriching for the protein (e.g. affinity chromatography, gel electrophoresis, and other biochemical methods), hydrolyzing the protein to amino acids, derivatizing the amino acids, and measuring the labeled amino acid by gas chromatography-mass spectrometry or gas chromatography-combustion-isotope ratio mass spectrometry (3, 4). More recently, proteomics methods have been developed that measure the labeled amino acid on the peptide level, eliminating the need for a protein enrichment step and enabling the monitoring of many proteins in a single experiment (5).Proteomics approaches to measuring protein turnover rates in mice have been accomplished by the introduction of a ¹⁵N stable isotope label. The labeled diets were created by supplementing a protein-free diet with a ¹⁵N enriched protein source. Price et al. (6) generated ¹⁵N-labeled protein from the alga, Spirulina platensis and Zhang et al. (7) introduced ¹⁵N-label in the form of lysate from the bacterium, Ralstonia eutropha. An advantage of using complete ¹⁵N labeling is the rapid incorporation of ¹⁵N and separation of isotope distributions between labeled and natural isotope abundance peptides, which reduces the need to deconvolute the two distributions. However, current methods require that the dietary protein content be derived from bacterial or alga lysate, a diet that is not normally fed to laboratory mice. As a result, measurements of protein turnover may not reflect conventional mouse model systems because of effects of diet on protein and amino acid metabolism. A more recent work by Claydon et al. (8) demonstrated a stable isotope labeling method by supplementing labeled valine into a standard mouse diet.The complex data generated from these analyses creates a data processing and analysis challenge; exemplified by recent software platforms that have been developed. Guan et al. (9) and Hoopmann et al. (10) demonstrated data analysis pipelines for ¹⁵N labeled SILAM and SILAC experiments. Here we describe the software platform, Topograph, we have developed for the analysis of liquid chromatography-tandem MS (LC-MS/MS) data from samples with isotopic labels. Topograph is able to deconvolute the complex spectra that may result from overlapping isotope distributions, regardless of the isotope label used. More uniquely, Topograph is able to calculate the relative isotope abundance (RIA)¹ of the amino acid precursor pool, which is necessary to correctly determine the amount of newly synthesized peptide and to subsequently calculate peptide and protein turnover rates.     

Intracuticular lipids of spinach leaves

The cuticular wax and cutin components of the cuticular membranes isolated from the leaves of two spinach cultivars have been determined. The membranes contain about 0·007 mg/cm² of cuticular wax which comprises monobasic acids (C₁₆–C₃₈) with hexadecanoic as the major component. The amounts of cutin are comparable with those of cuticular wax and the monomeric constituents are predominantly C₁₈ epoxy compounds. The most abundant monomer is 9,10-epoxy-18-hydroxyoctadecanoic acid (up to 63%) together with substantial amounts of 9,10,18-trihydroxyoctadecanoic acid (up to 22%). Also present are 9,10-epoxyoctadecane-1,18-dioic acid (6–7%) dihydroxyhexadecanoic acid (3–4%) and ω-hydroxymonobasic and fatty acid fractions. The tentative identification of two minor components, 18-hydroxyoxooctadecanoic and 9,10-epoxy-12,18-dihydroxyoctadecanoic acids, is also made. Although spinach membranes have a delicate structure their cutin composition is essentially similar to that of much more substantial membranes.     

Retarding effect of inhibitors of protein and RNA synthesis on chlorophyll and protein breakdown

Cycloheximide, ethionine,p-fluorophenylalanine, 6-azauracil, 5-diazouracil and vanillin, applied at relatively high concentrations, retarded the yellowing of kale (Brassica oleracea L. var.acephala) leaf discs in darkness, and stimulated it in light. All the compounds inhibited protein synthesis and retarded protein breakdown. Cycloheximide,p-fluorophenylalanine, diazouracil and vanillin also inhibited the incorporation of uracil-¹⁴C into RNA of senescing discs. Abscisic acid and 2-chloroethylphosphonic acid accelerated yellowing both in darkness and in light and stimulated the protein breakdown in senescing discs. Abscisic acid inhibited the chlorophyll, protein and RNA synthesis in detached, greening cucumber cotyledons. There was no direct correlation between the activity of a given compound as an inhibitor of yellowing in darkness, and the degree of inhibition of RNA synthesis. The arrest of yellowing in darkness is possibly a consequence of the retarded rate of protein breakdown. Yellowing in light, on the contrary, is dependent on the actual rate of protein synthesis.     

Cuticular permeance in relation to wax and cutin development along the growing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) leaf

The developing leaf three of barley provides an excellent model system for the direct determination of relationships between amounts of waxes and cutin and cuticular permeance. Permeance of the cuticle was assessed via the time-course of uptake of either toluidine blue or ¹⁴C-labelled benzoic acid ([¹⁴C] BA) along the length of the developing leaf. Toluidine blue uptake only occurred within the region 0–25 mm from the point of leaf insertion (POLI). Resistance—the inverse of permeance—to uptake of [¹⁴C] BA was determined for four leaf regions and was lowest in the region 10–20 mm above POLI. At 20–30 and 50–60 mm above POLI, it increased by factors of 6 and a further 32, respectively. Above the point of emergence of leaf three from the sheath of leaf two, which was 76–80 mm above POLI, resistance was as high as at 50–60 mm above POLI. GC-FID/MS analyses of wax and cutin showed that: (1) the initial seven fold increase in cuticular resistance coincided with increase in cutin coverage and appearance of waxes; (2) the second, larger and final increase in cuticle resistance was accompanied by an increase in wax coverage, whereas cutin coverage remained unchanged; (3) cutin deposition in barley leaf epidermis occurred in parallel with cell elongation, whereas deposition of significant amounts of wax commenced as cells ceased to elongate.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

Autotrophic synthesis of activated acetic acid from two CO2 in Methanobacterium thermoautotrophicum

In a previous study with Methanobacterium thermoautotrophicum evidence was presented that methanogenesis and autotrophic synthesis of activated acetic acid from CO₂ are linked processes. In this study one-carbon metabolism was investigated with growing cultures and in vitro.Serine was shown to be converted into glycine and activated formaldehyde, but only traces of label from [¹⁴C-3] of serine appeared in biosynthetic one-carbon positions. This seeming discrepancy could be explained if the same activated formaldehyde is an intermediate in biosynthesis and in methanogenesis from CO₂. This hypothesis was supported by demonstrating that [¹⁴C-3] of serine and [¹⁴C] formaldehyde were rapidly converted into methane, but a small portion of the label was also specifically incorporated into the methyl group of acetate. Methane and acetate synthesis in vitro were similarly stimulated by various compounds. These experiments indicate that the methyl of acetate and methane share common one-carbon precursor(s), i.e. methylene tetrahydromethanopterin, which can also be formed enzymatically from C-3 of serine or chemically from formaldehyde.Propyl iodide 20–40 M) and methyl iodide (1–3 M) completely inhibited growth in the dark. This effect was abolished by light. Methane formation was hardly affected. When ¹⁴CH₃I was applied at an only slightly inhibitory concentration, ¹⁴C was incorporated into the methyl of acetate. In vitro, similar effects on [¹⁴C] acetate formation from ¹⁴CO₂ or from [¹⁴C-3] of serine were observed, except that methyl iodide did not inhibit, but even stimulated acetate synthesis. These experiments indicate that a corrinoid is involved in acetate synthesis and probably not in methanogenesis from CO₂; the metal is light-reversibly alkylated and functions in methyl transfer to the acetate methyl.     

The effect of 2-thiouracil on RNA synthesis in pollen tubesof Nicotiana alata

The level of RNA in pollen is approximately 20 mg g^-1 and remains constant during 6 h pollen germinationin vitro also in the presence of 2-thiouracil which stimulates pollen tube elongation. The synthesis of RNA in pollen tubes was investigated according to the incorporation of the label from uracil-2-¹⁴C, 2-thiouracil-2-¹⁴C, orotic acid-5-³H, fructose-U-¹⁴C and from³²PO₄ ^3- into RNA fractions separated by methylated albumine kieselguhr chromatography. The distribution of radioactivity on elution profiles was different according to the radioactivity source, however it was not changed by the presence of 2-thiouracil in cultivation medium. 2-Thiouracil incorporates into pollen tube RNA at about 50% the rate of uracil. It inhibited the incorporation of orotic acid, of fructose and of phosphate into all RNA fractions. It is suggested that the analogue inhibits the enzymes involved in RNA synthesis essentially as 2-thiouridine-5’-phosphate.     

Effect of sweet cherry genes PaLACS2 and PaATT1 on cuticle deposition,composition and permeability in Arabidopsis

The cuticular membrane (CM) of sweet cherry (Prunus avium L.) fruit is severely strained during development. Strain results from a cessation of CM deposition during early development and is possibly caused by a downregulation of genes involved in CM synthesis. The objectives of our study were to investigate the effects of ectopic expression of two sweet cherry genes, PaLACS2 (a putative long-chain acyl-CoA synthetase) and PaATT1 (a putative cytochrome P450 monooxygenase), in Arabidopsis thaliana (L.). Effects on the expression of endogenous LACS2, ATT1 and LACS1 genes, wax and cutin composition, and cuticle permeability were investigated in 13 transgenic lines. Of these, six lines are selected for presentation based on the magnitude of the response. The amount of cutin increased in the PaLACS2 overexpression line C-L-29 and in the complemented lacs2-1 knockout mutant line l-L-14, but overexpression had no effect on cutin composition or wax. Wax deposition decreased in the complemented knockout lines l-L-14 and l-L-21. Overexpressing PaATT1 in A. thaliana line C-A-6 had no significant effect on cutin and wax deposition. In the complemented knockout lines a-A-7 and a-A-12, cutin deposition increased, whereas wax deposition was unaffected. The permeability of the cuticle for water and toluidine blue decreased in the PaLACS2 and PaATT1 complemented knockout lines. The results suggest that (1) PaLACS2 and PaATT1 expressed in A. thaliana are involved in cutin biosynthesis, and (2) their functions are consistent with those of a typical long-chain acyl-CoA synthetase (PaLACS2) and of a cytochrome P450 monooxygenase (PaATT1).     

Ureide Synthesis in a Cell-Free System from Cowpea (Vigna unguiculata [L.] Walp.) Nodules : STUDIES WITH O(2), pH, AND PURINE METABOLITES

The effect of O₂ and pH on the in vitro synthesis of ¹⁴C-labeled ureides from [8-¹⁴C]hypoxanthine in a cell-free system from cowpea nodules was investigated. Under conditions which suppressed uricase (EC 1.7.3.3) activity, namely low O₂ concentrations and low pH, ureide synthesis was inhibited and the ¹⁴C label incorporated into uric acid was increased. Conversely, conditions which increased uricase activity, namely high O₂ concentrations and high pH, also stimulated ureide synthesis, and the ¹⁴C label was incorporated principally into allantoin. The overall response of the system to O₂ concentration and pH indicated that the per cent distribution of total ¹⁴C label incorporated into uric acid was inversely related to that into allantoin. In the present study there was evidence that uricase (EC 1.7.3.3) controlled the in vitro rate of ureide synthesis in the cell-free system. Adenine and guanine inhibited xanthine dehydrogenase (EC 1.2.1.37) and as a consequence ureide synthesis from [8-¹⁴C]hypoxanthine was also inhibited.     

Transcriptional activity of heterocysts isolated from Anabaena variabilis

Nitrogenase activity, RNA synthesis, and protein synthesis were measured in heterocysts of Anabaena variabilis. Heterocysts labelled in situ for 4 h with [¹⁴C]uracil accumulated label in rRNA and tRNA to the same specific activity as RNA from vegetative cells. With isolated heterocysts, however, assimilation of [³H]uracil into RNa occurred at about 10% the rate in vegetative cells, and ceased 90 min after isolation. Pulse-chase experiments indicated that heterogeneous, high-molecular-weight RNA synthesized during the first 30 min of incubation was turned over during a 2 h chase, howver there was no accumulation of label in rRNA and tRNA as was seen with heterocysts labelled in situ and with vegetative cells. Assimilation of [³H]glycine into protein by isolated heterocysts was linear up to about 60 min, then proceeded at a slower rate for an additional 180 min. Maintenance of protein synsthesis and nitrogen fixation were both blocked by chloramphenicol and rifampicin. The data suggest that differentiated heterocysts continue to synthesize RNA and proteins and that these processes may contribute to the functional lifetime of heterocysts.     

Choline incorporation into phospholipids by microsomal fractions from L5178Y lymphoma

Microsomal fractions prepared from mouse lymphoma L5178Y grown in culture incorporated choline into cellular phospholipids when either choline or CDP-choline was used as the labeled precursor. Incorporation of label from CDP-choline was stimulated by Mg²⁺ and inhibited by Ca²⁺. In contrast, incorporation of label from choline required Ca²⁺ and was inhibited by EGTA. The characteristics of incorporation indicated that L5178Y cells have the capacity to utilize choline for phospholipid synthesis through both the Kennedy pathway and Ca²⁺-stimulated choline exchange.     

The effects of kinetin and contaminating bacteria on the incorporation of 32P-orthophosphate into various fractions of nucleic acid extracted from radish leaves

Summary By comparing ³²P-orthophosphate incorporation into nucleic acid extracts of sterile and non-sterile radish leaf discs, it was shown that contaminating bacteria cause a 2–4 fold increase in the rate of precursor incorporation and alter the pattern of label distribution after fractionation of the extracts by sedimentation through sucrose gradients or chromatography on MAK¹ columns. Using sterile senescing radish leaf discs, a stimulation of ³²P-orthophosphate incorporation into various fractions of nucleic acid was observed as a result of kinetin treatment.