Studies on organic acid metabolism and ethylene production during controlled atmosphere storage of apples {Mallus pumila MILLER, cv. Rolls)

Organic acid metabolism and ethylene formation during controlledatmosphere storage (CA-storage) of apples (Mallus pumila MILLER,cv Rolls) were studied. A higher titratable acidity was observedin apples during CA-storage as compared to those in air control.The incorporation of atmospheric ¹⁴CO₂ into malic acid was greaterin apples stored in the higher CO₂ concentration. The conversionof succinic acid-¹⁴C into fumaric acid-¹⁴C was slightly lessin the apple in modified high carbon dioxide atmosphere thanthose in air. O₂ uptake and CO₂ output by apple slices weremarkedly inhibited by the addition of succinic and malic acidsat a concentration higher than 25 mM. These factors seem to be the possible cause of a higher acidityof fruits stored in CA-condition. Ethylene production from wholefruits or tissue slices was markedly inhibited under CA-condition. The retardation of acid metabolism and the inhibition of ethyleneproduction of apples during CA-storage seem to be the importantfactors which help to maintain their storage quality. (Received March 18, 1970; )     

Influence of amino acids and organic antimetabolites on growth and biosynthesis of the chemoautotroph Thiobacillus neapolitanus strain C

Summary The growth of Thiobacillus neapolitanus strain C in liquid cultures was depressed by phenylalanine, p-fluorophenylalanine, cysteine, methionine, nor-leucine, azetidine-2-carboxylic acid, and chloramphenicol, but was little affected by glutamic acid, glycine, proline, azathymine, or oligomycin.Growing cultures assimilated ¹⁴C-labelled glycine, glutamic acid, phenylalanine, and tyrosine into protein. Tyrosine and phenylalamine were incorporated unchanged, but glutamate was used also for synthesis of arginine and proline. Glycine-¹⁴C contributed also to adenine and guanine synthesis. The extremely large amounts of phenylalanine incorporated into protein could indicate its toxicity to depend on its producing abnormal protein synthesis. Azetidine-2-carboxylic acid appeared to lower the amount of proline in the protein.Assimilation of glutamate and glycine by non-growing organisms was almost entirely dependent on energy from thiosulphate oxidation, thus suggesting a cause of obligate chemoautotrophy. Chloramphenicol specifically inhibited this thiosulphate-dependent incorporation of glutamate, glycine or CO₂ into protein at concentrations which did not affect total CO₂-fixation. Provided that energy is available from thiosulphate-oxidation this Thiobacillus is thus able to (a) activate exogenous amino acids; (b) incorporate them and CO₂ into protein by a chloramphenicol sensitive mechanism; (c) synthesise proline and arginine from glutamate; or adenine and guanine from glycine. Its biosynthesis thus depends on mechanisms like those of heterotrophs but requires to be driven by a chemolithotrophic energy supply.     

The Incorporation of 14C into the Protein of Particles Isolated from Plant Cells

The incorporation of ¹⁴C from labelled fructose, succinate,urea, and proline, by particulate preparations from dormantand tissue-cultured carrot cells, is examined. It is shown that¹⁴C is incorporated readily from proline, and less readily fromfructose. No significant incorporation occurs from succinateor urea. No differences are noted between the two kinds of preparation.It is concluded that the incorporation of ¹⁴C does not dependon prior transfer of the label to carbon dioxide followed byfixation of carbon dioxide, since the particles do not incorporate¹⁴C from supplied carbon dioxide. Incorporation of ¹⁴C by various fractions of dormant carrottissue is examined, and it is established that the greatestincorporation per mg. nitrogen occurs in particles isolatedat 10,000 g. A total cell homogenate fails completely to incorporate¹⁴C from proline into protein, and this may be due to suppressionof the activity of the particles by a constituent of the supernatantliquid. The presence of coconut milk reduces the incorporationof ¹⁴C from proline by particles sedimented at 10,000 g, andaddition of a protein hydrolysate reduces it further. Hydroxy-prolinedoes not appear to compete with proline for incorporation, andin this respect the paniculate preparations contrast with wholecells. Particles from carrot tissue are shown to be more active inincorporating ¹⁴C from proline than are particles extractedby the same procedure from red beet roots, potato tubers, andskunk cabbage inflorescences. They are, however, considerablyless active than a mitochondrial preparation from rat liver. It is demonstrated by paper chromatography that the bulk ofthe ¹⁴C incorporated in the particles from carrot cells remainsin proline and there is little or no conversion of proline tohydroxyproline in the preparations. The nature of the particlesemployed in this investigation is discussed, and their metabolismconsidered, in relation to the structure and activity of wholecells.     

Studies of Glycollate Utilization and Some Associated Enzymes of C1 Metabolism in the Endosperm of Ricinus communis L.

Glycollate metabolism in 5-day-old endosperm tissues of Ricinuscommunis L. was examined by feeding micromolar quantities of[2-¹⁴C]glycollate to tissue slices. It was found that glycollatecarbon was rapidly incorporated into glyoxylate, glycine, serine,and carbon dioxide. Only small amounts of ¹⁴C were incorporatedinto the sugars. Changes in the distribution of ¹⁴C with timesuggested that glyoxylate was a primary product and that glycineand serine were secondary products of glycollate metabolism.The results of feeding experiments are interpreted as indicatingthat a glycollate pathway leading to sugar biosynthesis is ofminor importance compared to the rapid utilization of glycollatefor the biosynthesis of glycine and serine. Enzymes necessaryto catalyse the incorporation of glycollate into glycine andserine have been examined in castor-bean endosperm extracts.These included: glycollic acid oxidase, gloxylic acid reductase,glyoxylate transaminase, N¹⁰ formyltetrahydrofolate synthetase,N⁵,N¹⁰-methylenetetrahydrofolate dehydrogenase, and serine hydroxymethyltransferase.     

Effect of chloramphenicol on ribonucleic acid synthesis in liver cells in suspension

1. Chloramphenicol has a stimulatory effect on the incorporation of radioactive phosphate into the RNA of perfused rat-liver slices, whole liver homogenates or the liver-cell suspensions, and no effect on the incorporation of [(14)C]adenine and [(14)C]uracil into the RNA of the tissue slices. 2. Chloramphenicol completely inhibits the incorporation of labelled adenine and uracil into the RNA of the cell suspensions, or into the RNA of homogenates derived from the whole liver tissues. 3. Chloramphenicol has at most a slight inhibitory effect on the transport of labelled adenine or uracil in the hepatic cells in suspension; in the slices, the transport of these bases is not inhibited at all. 4. The above observations indicate that: (a) unlike the tissue slices, hepatic cells in suspension are permeable to chloramphenicol; (b) in the presence of chloramphenicol, for reasons that are not clear, the conversion of the base into the appropriate nucleotide does not proceed.     

Metabolism of C1 unit precursors in Neurospora: Effects of media supplements on labelling of nucleic acids and protein

Mycclia of Neurospora crassa wild type (FE SC no. 853), harvestedduring the exponential phase of growth on defined minimal mediaincorporated glycine-2-¹⁴C, serine-3-¹⁴C and formate-¹⁴C intoproteins, DNA and RNA. Supplementing the growth medium with1 mM glycine increased the flow of glycine and formate carboninto these products. In contrast, this supplement decreasedthe incorporation of serine-¹⁴C. When such cultures were preincubatedfor 30 min with adenine, formaldehyde, formate or L-methionine,labelling of the nucleic acids and protein fractions by glycine-2-¹⁴Cwas altered. It is concluded that glycine increases the turnoverof C₁ units in Neurospora, resulting in greater contributionsof the C-2 in nucleic acid and protein synthesis. (Received May 14, 1977; )     

The Uptake of Amino-Acids and their Incorporation into the Proteins of Excised Barley Embryos

Methods are described by which barley embryos may be excisedand grown under sterile conditions on a medium containing sucrose,minerals, and a complete mixture of amino-acids. Growth underthese conditions was comparable with that of intact seedlingsand the uptake of sugar and amino-acid could be studied withoutdisturbing the metabolic steady state. Purified preparationsof the embryo proteins have been made and the constituent amino-acidsseparated. ¹⁴C labelling in these amino-acids was determinedby a new gas-scintillation method. In an isotopic competitionexperiment embryos were grown in ¹⁴C-sucrose with nitrate oran amino-acid mixture as nitrogen source. The presence of exogenousamino-acids suppressed the incorporation of carbon from carbohydrateinto amino-acid residues of the embryo protein. The degree ofsuppression varied, being undetectable for glutamic acid butalmost complete for lysine and leucine; it appeared to be relatedto the length of the synthetic pathway from carbohydrate tothe amino-acid. The evidence suggested that amino-acids areprotein precursors, and this conclusion was confirmed in furtherexperiments in which ¹⁴C-aspartic acid, -glutamine, -proline,-leucine, or -lysine were supplied singly in a complete mixtureof amino-acids. The ¹⁴C was found predominantly in the amino-acidresidue of protein corresponding to the ¹⁴C-amino-acid supplied,with smaller amounts in other amino-acids of the same or relatedfamilies. Aspartic acid and glutamine yielded appreciable quantitiesof respiratory carbon dioxide, although the contribution wassmall compared to that of sucrose. Little carbon was lost ascarbon dioxide from leucine or lysine. The results are discussedin relation to the role of amino-acids in protein synthesis,and to the existence of feedback control in the amino-acid metabolismof higher plants.     

Oleic acid synthesis in the fruit of Persea americana MILL

The tissue slices from the mesocarp of avocado could incorporateradioactive acetate into lipids. Oleic, palmitic and stearicacids were the most labeled fatty acids found to accumulatein triglycerides. The conclusions that oleic acid was formedby way of chain elongation of already unsaturated short-chainfatty acids, and that there was no evidence for the desaturationof stearic acid were based on the following observations: 1)Stearic acid-¹⁴C was incorporated into triglycerides by thetissue slices without formation of oleic acid. 2) The oleicacid synthesized from specifically labeled acetate was not randomlylabeled. The specific radioactivity of azelaic acid moiety ofoleic acid was rapidly increased while that of pelargonic acidmoiety was gradually increased. 3) An unexpected rise of stearicacid was observed among commonly occurring fatty acids in thetissue slices. This was accentuated by anaerobiosis which prevailedduring vacuum infiltration of labeled acetate. ¹Present address: Department of Botany, Faculty of Agriculture,Hokkaido University, Sapporo, Japan. (Received January 13, 1969; )     

Isolation of 1-O-trans-p-Coumaroyl-r{beta}-D-glucopyranose from Sweet Potato Roots and Examination of Its Role in Chlorogenic Acid Biosynthesis

1-O-trans-p-Coumaroyl-rß-D-glucopyranose (p-coumaroyl-D-glucose)was isolated from slices of sweet potato root which had beenincubated with trans-cinnamic acid. Pre-loaded trans-cinnamicacid efficiently trapped the radioactivity from L-[U-¹⁴C]-phenylalanineand reduced its incorporation into chlorogenic acid by 75% ofcontrol values in disks of sweet potato root. In the root diskssupplied with trans-[3-¹⁴C]-cinnamic acid, the radioactivitywas transferred first to trans-cinnamoyl- D-glucose, then top-coumaroyl-D-glucose, and subsequently to chlorogenic acidand isochlorogenic acid. These results support our earlier propositionthat p-coumaroyl-D-glucose is involved in the biosynthesis ofchlorogenic acid as an intermediate adjacent in the pathwayto trans-cinnamoyl-D-glucose in sweet potato roots. (Received April 11, 1988; Accepted August 9, 1988)     

Alicyclic acid metabolism in plants 7. Metabolism of 14G-labelled alicyclic acids in germinating Phaseolus mungo seeds

When Phaseolus mungo seeds were allowed to germinate in 5–20mM quinate solution in the dark, a marked increase in the endogenousshikimic acid level occurred in their tissues. The acid levelrose distincdy on the 2nd day of germination and reached a maximumon the 4th day. The quinate-¹⁴C fed to germinating seeds waspredominantly converted to shikimic acid, and little radioactivitywas found in 3-dehydroshikimic acid. When quinate-¹⁴C was suppliedsimultaneously with relatively high concentrations of 3-dehydroquinicacid or 3-dehydroshikimic acid, its conversionto shikimic acidwas restrained, but hardly any radioactivity was trapped ineither of the dehydro compounds. 3-Dehydroquinic acid-¹⁴C or3-dehydroshikimic acid-¹⁴C fed to the seeds was metabolizedpreferentially to shikimic acid. The experimental results arediscussed with respect to the metabolic relationship betweenquinic acid and odier alicyclic acids in the aromatic biosynthesisof P. mungo seedlings. (Received October 16, 1975; )     

Effect of Ethrel and Ceratocystis fimbriata on the Synthesis of Fatty Acids and 6-Methoxy Mellein in Carrot Root

The rate of incorporation of ¹⁴C from acetate-1-¹⁴C into fatty acids by carrot root discs, 18 hours after inoculation with Ceratocystis fimbriata, was 9-fold greater than that in freshly cut discs. The rate in discs treated with water or Ethrel was 3-fold greater. The rate of incorporation of ¹⁴C from glucose-U-¹³C into fatty acids was 3-fold greater 18 hours after any of the above treatments. The rate of ¹⁴C incorporation from malonate-2-¹⁴C into fatty acids 24 hours after inoculation with C. fimbriata or treatment with water was 25 and 60%, respectively, of that in freshly cut discs. Linoleic acid was the principal fatty acid in carrot root, but incorporation of ¹⁴C from acetate-1-¹⁴C into the acid was low until 18 hours after inoculation with C. fimbriata or treatment with Ethrel. Turnover rates of the fatty acids appeared low and were similar for all treatments.     

PHOTOSYNTHESIS OF GLYCINE AND SERINE IN GREEN PLANTS

1. The effects of "carbonyl" reagents on the photosyntheticin-corporation of ¹⁴CO₂ into the assimilation products of tobaccoand spinach leaves were studied. The presence of "carbonyl"reagents causes an increase in the ratio of ¹⁴CO₂ incorporatedin glycine and a decrease in serine. The incorporation of ¹⁴Cfrom glycolate-1-¹⁴C and glycolaldehyde-2-¹⁴C into glycine andserine was also affected by "carbonyl" reagents, as in the caseof ¹⁴CO₂-experiment. 2. The feeding experiments of glycine-1-¹⁴C and serine-1-¹⁴Cin the presence and in the absence of "carbonyl" reagents revealedthat these reagents inhibit the conversion of glycine to serine. 3. The results obtained above, together with the effects ofthiols on ¹⁴CO₂ incorporation presented in this paper, supportthe assumption that glycine and serine are formed via glycolateand glyoxylate during photosynthesis in green plants. 4. Comparison of ¹⁴C incorporation in malate from ¹⁴CO₂, glycolate-1-¹⁴C,glycine-1-¹⁴C and serine-1-¹⁴C in the presence and in the absenceof "carbonyl" reagents suggested the occurrence of the pathwayof the malate formation via glycolate and glyoxylate, not passingthrough glycine and serine, during photosynthesis. ¹ A part of this paper was presented at the Symposium on "Nitrogenand Plant" by the Japanese Society of Plant Physiologists, inOctober, 1963 ² Present address: Radiation Center of Osaka Prefecture, Sakai,Osaka     

Myxospore coat synthesis in Myxococcus xanthus: in vivo incorporation of acetate and glycine.

Myxospore coat synthesis in Myxococcus xanthus was studied by incorporation of [(14)C]acetate into intermediates in the biosynthesis of coat polysaccharide and into acid-insoluble material during vegetative growth and after glycerol induction of myxospores. During short labeling periods at 27 degrees C, the radioactivity was shown to be located primarily in N-acetyl groups rather than sugar moieties. Two hours after glycerol induction, the pools of N-acetylglucosamine 6-phosphate and uridine 5'-diphosphate-N-acetylgalactosamine (UDPGalNAc) plus uridine 5'-diphosphate-N-glucosamine increased about twofold and were labeled at twice the rate measured for vegetative cells. The increased rate of synthesis of UDPGalNAc and its precursors could be correlated with increased enzyme activities measured in vitro. Controlled acid hydrolysis revealed that the galactosamine portion of the myxospore coat was N-acetylated. After glycerol induction, the incorporation of acetate into acid-insoluble material increased threefold. This enhanced incorporation was sensitive to neither penicillin nor d-cycloserine. In contrast, bacitracin inhibited the incorporation of [(14)C]acetate into acid-insoluble material more effectively 2 h after myxospore induction than during vegetative growth. Chloramphenicol added to cells 90 min after induction blocked further increase in the rate of [(14)C]acetate incorporation. Since the myxospore coat contains glycine, polymer synthesis was also measured by chloramphenicol-insensitive [(14)C]glycine incorporation into acid-insoluble material. Although protein synthesis decreased after glycerol induction, glycine incorporation increased. Two hours after induction, glycine incorporation was only 75% inhibited by chloramphenicol and rifampin. The chloramphenicol-insensitive rate of incorporation of [(14)C]glycine increased during the first hour after myxospore induction and reached a peak rate after 2 to 3 h. The chloramphenicol-resistant incorporation of [(14)C]glycine was resistant to penicillin but sensitive to bacitracin.     

Conversion of L-Ascorbic Acid to L-Idonic Acid, L-Idono-{gamma}-lactone ane 2-Keto-L-idonic Acid in Slices of Immature Grapes

L-[1-¹⁴C] Ascorbic acid ([1-¹⁴C]AA) was vacuum-infiltrated intoslices of immature grapes (Vitis labrusca L., cv. "Delaware")after which incorporation of ¹⁴C into each metabolite was investigated.Under the experimental conditions used, the metabolic reactionof AA proceeded at a constant rate within the reaction period(5 hr). As a result, ¹⁴C first appeared in three metabolic products;L-idonic acid, L-idono--lactone and 2-keto-L-idonic acid (=2-keto-L-gulonicacid), after which incorporation of ¹⁴C into tartaric acid (TA)took place. When slices of grapes were treated with iodoaceticacid, the incorporation of ¹⁴C into TA completely stopped andin the inhibitor experiment, the amount of ¹⁴C which had beenincorporated into TA in the control was found to be dividedamong L-idonic acid, L-idono--lactone and 2-keto-L-idonic acid.These results are strong evidence that at least one of thesethree compounds is the effective precursor of TA and is locatedon the metabolic pathway between AA and TA in the grape. (Received December 3, 1981; Accepted February 10, 1982)     

The Incorporation of 14C-Proline into the Proteins of Growing Cells: II. A NOTE ON EVIDENCE FROM CULTURED CARROT EXPLANTS BY ACRYLAMIDE GEL ELECTROPHORESIS

The method of acrylamide gel electrophoresis has been appliedto the separation of the proteins of growing carrot explantswhich are soluble in a buffer solution at pH 8.3. At least ninedistinct bands were detectable in this way. When carrot tissuehad absorbed ¹⁴C-proline it entered into the composition ofall these bands and, in all except one, it was converted tohydroxyproline to different degrees which characterized theband in question. Thus, in the soluble protein the ¹⁴C-hydroxyproline:¹⁴C-proline ratio varied from 0 to 0.53. The bulk of the proteinin the tissue was insoluble in buffer at pH 8.3; it containedthe bulk of the radioactivity absorbed from proline (84.8 percent.); and its average ¹⁴C-hydroxyproline : ¹⁴C-proline ratiowas the highest of all (1.28). A particulate protein preparation,separated at 25,000 g. from the soluble protein, had an intermediateratio (0.643) of ¹⁴C-hydroxyproline to ¹⁴C-proline. Therefore,there are in cultured carrot explants many distinct proteinmoieties which incorporate ¹⁴C-proline, and they convert itto ¹⁴C-hydroxyproline to very different degrees. The evidenceis consistent with the incorporation of the proline, first intothe various soluble proteins which are electrophoretically separableand subsequently, with progressively greater hydroxylation,into the more insoluble protein that constitutes the bulk ofthe protein of the cell and its organelles. It is, therefore,quite incorrect to say (as some have done) that all of the hydroxyproline-containingprotein is in very close association with the cell wall, forpart of it is present in the cell in soluble and electrophoreticallyseparable forms.     

ROLE OF NUCLEIC ACID AND PROTEIN METABOLISM IN THE INITIATION OF GROWTH AT GERMINATION

When dry decotyledonized embryos of Raphanus are supplied withwater, a brief period of water absorption (phase A) is followedby a period of no fresh weight increase (phase B) which lastsfor 8 hr at 30°. In this period, embryos become ready toadvance into the period of fresh weight increase (phase C). When embryos were exposed to various concentrations of thiouracilor actinomycin D solution from 0 hr of water supply, increasesin fresh weight and in RNA content measured at 13 hr were inhibitedin parallel with each other. Chloramphenicol and puromycin inhibitedthe fresh weight increase without affecting the RNA increase.When embryos were exposed to thiouracil or puromycin for 2,4 and 6 hr, beginning at 0 hr of water supply, the start ofphase C delayed 2, 4 and 6 hr, respectively. When these drugswere given after phase B had progressed at least for 2 hr, thedelay of the start of phase C was shorter than the period ofthe drug treatment. If given at the end of phase B, thiouraciland actinomycin D inhibited the incorporation of ¹⁴C-uracilbut not the fresh weight increase, while chloramphenicol andpuromycin inhibited the latter without inhibiting the former. During phase B, protein content per dry weight of embryo didnot increase, but the rate of ¹⁴C-leucine incorporation increasedremarkably to reach the level in phase C. Incorporation of labeledleucine was inhibited if embryos were subjected to thiouracilor actinomycin D action during phase B, but not if the drugswere given when phase B had been completed. Puromycin and chloramphenicolinhibited the incorporation whenever they were given. The increase in respiratory activity during phase B was inhibitedrelatively little by the above mentioned four drugs. In conclusion syntheses of RNA and protein seem to be essentialfor the progress of phases B and C, protein synthesis havinga more direct effect. (Received September 17, 1965; )     

Root Development and Source-Sink Relations in Carrot, Daucus carota L: II. EFFECTS OF ROOT PRUNING ON CARBON ASSIMILATION AND THE PARTITIONING OF ASSIMILATES

Physiological responses to root pruning were investigated bycomparing ¹⁴CO₂ fixation rates, the partitioning of ¹⁴C-labelledassimilate, and soluble and insoluble carbohydrate levels inthe leaves of carrot plants following the removal of some ofthe fibrous roots, or fibrous roots and part of the tap root.Root pruning reduced ¹⁴CO₂ fixation by 28–45% but leafspecific activity (¹⁴C assimilation g-¹ leaf fresh weight) wasunchanged. The proportion of total assimilate exported to theroot system increased following root pruning and this was atthe expense of the developing leaves. In younger plants (wherethe tap root received 10% of the assimilate) the supply of ¹⁴Cto the tap root was maintained in spite of root pruning. However,shortening the tap root to 3 cm in older plants (in which 30%of the fixed 14C was normally exported to the developing storageorgan), reduced its sink capacity and resulted in slightly greaterretention of ¹⁴C in the mature leaves. Greater concentrationsof insoluble carbohydrate were found in the mature leaves followingroot pruning but soluble sugar content was unaffected. Onlysmall differences were observed in the distribution of ¹⁴C betweensoluble and insoluble carbohydrate fractions when plants werefed ¹⁴CO₂ several days after the root pruning operations. Thesephysiological responses were mainly associated with the removalof fibrous roots and support the view that the fibrous rootsystem is more important than the developing storage organ inregulating growth in young carrot plants.     

Proline Metabolism in Leishmania donovani Promastigotes*

Oxygen uptake of Leishmania donovani culture promastigotes was stimulated by L-proline and to a lesser extent by L-glutamate and L-arginine. L-proline reversed partially KCN-induced inhibition of respiration and completely, inhibition caused by malonate. Labeled proline, glutamate, alanine, and arginine were detected by thin layer chromatography in the free amino acid pool from cells incubated with L-proline-¹⁴C. Labeled tricarboxylic acid cycle intermediates, α-ketoglutarate, succinate, fumarate, malate, and oxaloacetate, also were found by this method in extracts from organisms incubated with L-proline-¹⁴C which contained also pyruvate. Cells incubated with malic acid-¹⁴C contained labeled alanine, glutamate, and arginine. Labeled L-proline was not found in promastigotes incubated with D-glucose-¹⁴C, although arginine, glutamate, and alanine were detected in extracts from these organisms. Indirect evidence for the presence of a NADP-dependent malic enzyme was obtained by Ochoa's method. All results suggest the presence of a proline-glutamate interconversion pathway in L. donovani promastigote culture forms.     

EFFECTS OF CHLORAMPHENICOL AND KINETIN ON UPTAKE AND INCORPORATION OF AMINO ACIDS BY TOBACCO LEAF DISKS

The effects of chloramphenicol and kinetin on uptake and incorporationof ³⁵S-methionine and some ₁₄C-amino acids have been investigatedin leaf-disks of Nicotiana rustica in light and dark. Chloramphenicolin a concentration of 1 mg per ml inhibits the uptake of aminoacids from 30 to 60 per cent compared with the water control.The incorporation of amino acids into bulk protein is stronglyinhibited in light (40 to 70 per cent), but only to a smalldegree in dark (10 to 20 per cent), as revealed also by ¹⁴CO₂-photosynthesisof the disks and following treatment with chloramphenicol indark. The stimulating effect of kinetin on uptake and incorporationof amino acids is dependent upon its concentration (10^–5to 10^–6 M ; but 10^–4 M solution inhibits stronglyboth uptake and incorporation). The stimulation seems to influencemore incorporation than uptake processes. Possible interactionsof chloramphenicol and kinetin in the protein metabolism oftobacco leaves have been discussed. (Received April 27, 1964; )     

The Induced Resistance Response of Carrot Root Slices to Heat-killed Conidia and Cell-free Germination Fluid of Botrytis cinerea Pers. ex Pers.: 2. Nuclear Migration, Nucleolar Volume and Uptake of Tritiated Uracil

Sixty-eight per cent of nuclei in the cells of the upper fourlayers of carrot slices treated with heat-killed conidia ofBotrytis cinerea for 6 h followed by inoculation with live sporesfor 18 h, migrated to the cell face nearest to the treated surface,compared with 46 per cent in cells of control slices showinga wound-healing response only. Nucleolar volumes in the surfacecell layers of control slices increased from a mean of 1.0 µm³to 3.8 µm³ over 24 h, and in ‘induced’ slicesto 7.28 µm³. Using a 40 min pulse of [5–³H]uracil,there was an increase within 15 h of slicing in the number oflabelled nuclei in cells from control slices undergoing healing.Within 8 h after treatment of slice surfaces with heat-killedconidia, there was an accelerated incorporation of label into‘nuclear’ RNA. Slices from roots cold-stored for12 months failed to show an induction response and nucleolarvolumes did not increase more than in control slices. Theseresults are discussed in relation to active defence mechanismsin plant tissue. Botrytis cinerea, carrot, induced resistance, nuclear migration, nucleolar volume, RNA incorporation