The Incorporation of 14C-Proline into the Proteins of Growing Cells: I. EVIDENCE OF SYNTHESIS IN DIFFERENT PROTEINS AND CELLULAR COMPONENTS

¹⁴C-proline was supplied to aerated potato disks, in which celldivision was occurring, and also to rapidly growing potato carrotexplants. It was absorbed and incorporated into all the subcellularprotein fractions examined, including the electrophoreticallydistinguishable fractions of the soluble protein of the potatodisks and explants. The ¹⁴C-proline was partially convertedto ¹⁴C- hydroxyproline in all the protein fractions, exceptfor one of the soluble protein fractions of potato explantsand the soluble proteins of one set of potato disks. Most ofthe ¹⁴C-proline and ¹⁴C-hydroxyproline contained in the tissuewas found in the soluble protein and also in the cellular fragmentsobtained by centrifugation at 500 g. The relative importanceof the soluble protein in the incorporation of ¹⁴C-proline andits conversion to ¹⁴C-hydroxyproline was greatest over a shortperiod of a few hours of contact with the ¹⁴C-proline supplied.Over a longer period (70 hours) the cellular fragments (500g) had become the most important and contained over 40 per cent.of the total ¹⁴C, and more than 60 per cent. of the ¹⁴C-hydroxyproline,in the protein of the tissues. In the soluble fraction of potatoexplants, seven protein bands were distinguishable on electrophoresis.A different but characteristic value of the ratio ¹⁴C-hydroxyprolineto ¹⁴C-proline was associated with each protein band, exceptfor the one region where ¹⁴C-hydroxyproline did not occur. Thebasic proteins (i.e. those moving towards the cathode) werethe most active in the incorporation of ¹⁴C-proline and itsconversion to ¹⁴C-hydroxyproline. The rather general distributionof the ¹⁴C-hydroxyproline is noted and the possible siginificanceof the basic proteins and the proteins associated with the cellularfragments (500 g) is considered in relation to the growth, celldivision, and cell wall formations which occurs in the rapidlygrowing tissue cultures.     

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.     

The Use of C14-Proline by Growing Cell: Its Conversion to Protein and Hydroxyproline

C¹⁴-proline is readily absorbed by growing tissue cultures ofcarrot root phloem and of potato tuber in experiments carriedout under aseptic conditions. The C¹⁴-proline rapidly entersinto the protein of the tissue, appearing there in as shorta period as 15 minutes, and, thereafter, the amount incorporatedinto the protein bears a linear relation to time. Virtuallyall the C¹⁴ appears in the protein hydrolysate in the form ofproline and hydroxyproline. It is shown that the conversionfrom proline to hydroxyproline occurs after the C¹⁴-prolineis combined into the protein and that this conversion proceedsprogressively with time. The ratio of C¹⁴ as proline to C¹⁴as hydroxyproline declined progressively from a value of 4.0after 30 minutes of contact and seemed to become stabilizedeventually at 0.7. C¹⁴-hydroxyproline, which can be absorbedby the tissue, seems not to be incorporated into the proteinas such. The protein moiety which contains the C¹⁴-hydroxyprolinefrom C¹⁴-proline represents a stable protein which is not metabolizedand whose carbon does not ‘turn over’. This inertprotein seems to be characteristic of cells which are in rapiddivision under the influence of coconut milk or are synthesizingprotein in response to other stimuli such as the events at acut tissue surface. The protein in question seems to be presentmainly in the cytoplasm rather than in its paniculate inclusions.These results are compatible with earlier views which requirethat part of the protein in the cell ‘turns over’its carbon, whereas another part does not do so.     

Variation in 14C Partitioning in the Tips of Growing Stolons of Potato (Solanum tuberosum L.)

¹⁴C partitioning was examined in growing stolons of field-grownpotato (Solanum tuberosum L.) cv. Maris Piper. Considerablevariation was evident on single plants and on a fresh weightbasis many stolon tips, which showed no signs of sub-apicalswelling, had higher specific activities (cpm g^–1 f. wt)of ¹⁴C in both ethanol soluble and insoluble forms than larger,visibly tuberized stolons. Furthermore, many tips of low freshweight had a higher insoluble to soluble ¹⁴C ratio than visiblytuberized stolons suggesting greater efficiency of conversionof soluble ¹⁴C to insoluble ¹⁴C in the smaller stolons. Theresults suggest that the onset of visible ‘tuberization’,namely the sub-apical swelling of the stolon, is preceded byincreased soluble carbon accumulation at the stolon tip togetherwith an increase in the conversion of soluble to insoluble formsof carbon. Tuberization, ¹⁴C, stolon tip     

Early Replication of Highly Repeated DNA Sequences during Dedifferentiation of Carrot

When carrot explants are placed on an agar medium containingphytohormones, a satellite DNA begins to replicate earlier thanthe bulk DNA during the first cell division cycle. The majorityof this early replicating satellite DNA was previously shownto have an identical buoyant density to ribosomal DNA (rDNA)(Hase et al. 1982). Molecular sizes of EcoRI-digests of ³H-labeledDNA were analyzed in the present study by gel electrophoresisfollowed by fluorography. Most of the labeled DNA bands didnot correspond to EcoRI-digests of carrot rDNA. The resultsindicated that the majority of the early replicating satelliteDNA is not rDNA, but probably a type or types of highly repeatedDNA sequences. (Received June 6, 1985; Accepted November 4, 1985)     

Storage Pools and Turnover Systems in Growing and Non-growing Cells: Experiments with 14C-Sucrose, 14C-Glutamine, and 14C-Asparagine

¹⁴C-labelled sucrose, glutamine, and asparagine have been suppliedto aseptically cultured carrot explants that either grew rapidlyby cell division or, by contrast, only slowly by cell expansion.The radioactive substrates were supplied in a brief ‘pulse’followed by a much longer period during which the tissues weresupplied with ¹²C-substrates. The passage of ¹⁴C through thevarious soluble compounds of the tissue and into the proteinwas followed. Alternatively, the ¹⁴C-labelled compound was suppliedthroughout the entire period of an experiment while the tissuealso received ¹²C-sucrose. The pulse-labelling experiments demonstrateturnover and the fate of the breakdown products, as well asthe emphasis placed on this kind of metabolism by cells at differentlevels of activity in their growth. The long-term labellingexperiments show the different ways in which carbon from varioussources may be used and how these pathways are affected by growth.The amount of ¹⁴C present in the various free (ethanol soluble)and combined (ethanol insoluble, acid-hydrolysable compounds—proteins)was determined, as well as the specific activity of the carbonin each compound. The fate of ¹⁴C supplied as sucrose had muchin common with ¹⁴C supplied as glutamine, with respect to theease with which it entered both the protein being synthesizedand the carbon dioxide evolved, but it was very different from¹⁴C supplied as asparagine. To interpret these data, compartmentsor pools of metabolites are postulated in the organized cell;exogenous ¹⁴C-sucrose and ¹⁴C-glutamine readily furnish carbonfor pools of amino-acids en route to protein, which are protectedfrom both the stored compounds and those which arise after proteinbreakdown. However, exogenous ¹⁴C-asparagine enters, is accumulated,and persists in the pool of stored compounds which also receivethe nitrogen-rich substances that arise from protein breakdown.The kinetic data and the specific activities of the carbon inits various forms require that protein breakdown and re-synthesisoccur concomitantly, that the stimulus to grow, exerted by coconutmilk, accentuates protein synthesis and also the pace of itsturnover, that some respired carbon dioxide arises from protein,and that this moiety of the respiration is increased by thecoconut-milk stimulus as it accentuates the pace of cyclicalturnover. In similar experiments with free cells from differentplants, the same general conclusions apply, but the rates ofturnover of protein are greater in free cells than in tissueexplants. Some specific differences, however, exist. Cells ofArachis, the only legume investigated, permit ¹⁴C-asparagineto contribute, like ¹⁴C-glutamine, to both protein synthesisand respired ¹⁴CO₂; it is not merely segregated in a storagepool. Thus, by virtue of their organization, plant cells maintainthe same substances simultaneously in distinct phases or compartments,where they play distinctive roles, without mingling. Geneticsendows each cell with the information that makes its biochemicalreactions feasible; the organization of the cells determineshow far the feasible becomes practised in cells in any givensituation.     

Cell Wall Metabolism in Developing Strawberry Fruits

Cell wall metabolism was studied in strawberry receptacles (Fragariaananassa, Duchesne) of known age in relation to petal fall (PF).Polysaccharide and protein composition, incorporation of [¹⁴C]glucoseand [¹⁴C]proline by excised tissue, and the fate of ¹⁴CO₂ fixedby young, attached fruits were followed in relation to celldivision, cell expansion, fine structure, and ethylene synthesis. Cell division continued for about 7 d after PF although vacuolationof cells was already beginning at PF and the subsequent cellexpansion was logarithmic. There was an associated logarithmicincrease in sugar content per cell and a decreasing rate ofethylene production per unit fresh weight. During cell expansion radioactivity from [¹⁴C]glucose was incorporatedinto fractions identified as starch and soluble polyuronideand into glucose and galactose residues in the cell wall. Radioactivityfrom [¹⁴C]proline was also incorporated into the cell wall,but only 10 per cent of this activity was found in hydroxyproline.Correspondingly wall protein contained a low proportion of hydroxyprolineresidues. The proportion of radioactivity from ¹⁴CO₂ fixed byfruitlets remained constant in most sugar residues in the cellwall. The proportion of radioactivity in galactose fell, indicatingturnover of these residues. Between 21 and 28 d after PF receptacles became red and softenedbut there was no change in the rate of ethylene production.Cell expansion continued for at least 28 d. Tubular proliferationof the tonoplast and hydration of middle lamella and wall matrixmaterial had begun 7–14 d after PF but became extremeduring ripening. Associated with the hydration of the wall,over 70 per cent of the polyuronide in the wall became freelysoluble, and arabinose and galactose residues lost from thewall appeared in soluble fractions. There was no increase intotal polysaccharide during ripening and incorporation of [¹⁴C]glucoseinto polysaccharides ceased, although protein increased andincorporation of [¹⁴C]proline into wall protein continued.     

Myo-inositol Biosynthesis and Galactose Utilization by Wild Carrot (Daucus carota L. var. carota) Suspension Cultures

Wild carrot (Daucus carota var. carota) cell suspensions (63–120µm in diameter) were grown on a mineral salt medium containingdifferent carbon sources in the presence (10 mM) and absenceof myo-inositol. The data obtained after 14 and 21 days of growthshow that an external supply of myo-inositol is not essentialfor growth and development of wild carrot embryos. A linearrelationship was found between growth (d. wt) and embryo numberin the presence and absence of myo-inositol. Standard stock cell suspensions never exposed to exogenous myo-inositoland grown in the absence of 2, 4-D with glucose or galactoseas the carbon source synthesized radioactive myo-inositol whenexposed to D-[1–¹⁴C]glucose or D-[1–¹⁴C]galactose.Gas chromatographic analyses revealed the presence of myo-inositolin the bulk tissue grown in the presence of 2.25 µM 2,4-D with glucose, galactose, fructose or mannose as the solecarbohydrate. We could not detect any component indicating anisomer or a methylated derivative of an inositol in the tissueextracts. Stock cultures were maintained (with 2, 4-D) successfully forat least three successive sub-cultures on D-galactose as thesole carbohydrate. The growth achieved over this culture periodshowed that wild carrot cells used by us could quickly adaptto grow on D-galactose as rapidly as they grow on sucrose. Daucus carota L., wild carrot, suspension cultures, myo-inositol, galactose     

Long-term Metabolism of [14C]Sugars in Stored Potatoes

[¹⁴C]Sucrose, [¹⁴C]glucose and [¹⁴C]fructose were introducedinto potato tubers held at 10 °C and the redistributionof label chased over a 65 d period in storage. Respiratory losseswere identical in all treatments, as was the partitioning of¹⁴C between soluble and insoluble forms. Sucrose was the predominantlabelled sugar in the tubers after 20 h, regardless of the original[¹⁴C]sugar introduced, and was loaded and distributed throughoutthe tubers by the internal phloem system. After 20 h the proportionsof labelled sugars bore no relationship to those of the unlabelledendogenous sugars. However, with time the percentage of ¹⁴Cin sucrose fell while that in glucose increased and by 65 dthe proportions of the labelled sugars more closely resembledthe endogenous pools. Fructose represented a consistently lowproportion of both the labelled and unlabelled sugars. By 21d a considerable proportion of the soluble ¹⁴C had been convertedto starch (approx. 25% of the total tuber 14C), this value remainingrelatively constant for the remainder of the storage period.Sprouts which formed on the tubers contained up to 6% of thetotal tuber ¹⁴C but less than 0.2% of the tuber dry matter.It is suggested that the bulk of the translocated [¹⁴C]sucroseentered the symplast and exchanged slowly with the bulk of thesugars in the storage cell vacuoles. [¹⁴C]sugars, phloem loading, starch, potato tuber, Solunum tuberosum, cold storage     

Salinity Stress and the Content of Proline in Roots of Pisum sativum and Tamarix tetragyna

Amino acid composition of the free amino acid pool and the TCA-insolubleprotein fraction were investigated in root tips of pea and Tamarixtetragyna plants grown at various levels of NaCl salinity. Salinitystress induced an increase of proline content, mainly in thefree amino acid pool in both plants, and of proline or hydroxyprolinecontent in the protein. Externally-supplied proline was absorbedand incorporated into protein, by pea roots, more effectivelythan by Tamarix roots. Salinity stress, apparently, stimulatedthe metabolism of externally-supplied labelled proline. Pearoots have a very large pool of free glutamic acid; however,70 per cent of the ¹⁴C from externally-supplied ¹⁴C-U-glutamicacid was released as CO₂. Very small amounts of it were incorporatedinto protein. No measurable amount of radioactivity could bedetected in any one of the individual amino acids, either ofprotein hydrolysate or the free amino acid pool. Proline very effectively counteracted the inhibitory effectof NaCl on pea seed germination and root growth. A similar effectbut to a lesser degree was achieved with phenylalanine and asparticacid. The feasibility of proline being a cytoplasmic osmoticumis discussed.     

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.     

Increased sensitivity of scleroderma fibroblasts in culture to stimulation of protein and collagen synthesis by serum

Serum effects on ¹⁴C-proline incorporation and ¹⁴C-hydroxyproline synthesis by normal and scleroderma fibroblasts in culture were studied. Serum resulted in 97% and 212% increases in ¹⁴C-proline incorporation in two lines of scleroderma fibroblasts while the increase in normal fibroblasts was only 53%. Effects on collagen synthesis were more pronounced. Addition of serum resulted in 124% and 445% increments in ¹⁴C-hydroxyproline synthesis in the scleroderma fibroblasts but only a 43% increment in the normal fibroblasts. The results indicate that cultured scleroderma fibroblasts have increased sensitivity to biosynthetic stimulation by serum and this mechanism may be of pathogenetic importance in the excessive collagen accumulation characteristic of the disease.     

Studies on the distribution of 14C-malformin A in major fractions of Phaseolus vulgaris L.

The distribution pattern of ¹⁴C-malformin in major fractionsof Phaseolus vulgaris L. seedlings shifted during water treatmentin the absence of malformin. From these shifts, and by comparisonof the ¹⁴C distribution patterns at the base and top of theseedlings, it was concluded that some ¹⁴C-malformin enters thecell and proceeds to the cell wall via intermediate compounds.As a working hypothesis it was suggested that in roots ¹⁴C-malforminfirst appears in a soluble "small molecules" fraction, bindsto a soluble protein fraction, and proceeds via die wall lipidfraction to the wall itself. Direct binding of some ¹⁴C-malforminto the wall fraction was not precluded. In leaves, the pathwayof ¹⁴C-malformin to the cell wall was similar in some respectsto that in roots. ¹ Present address: American Cyanamid, P.O. Box 400, Princeton,New Jersey 08540, U.S.A. (Received January 21, 1976; )     

The Effect of Abscisic Acid on Amino Nitrogen and Protein Content of Potato Plants in Relation to the Inhibition of Nitrate Reductase Activity

Treatment of potato plants grown in nutrient solution with 3.8µM ABA resulted in reduced soluble protein in roots andin leaves at 24 h, but not in stems. This treatment reducedin vivo nitrate reductase activity in all organs for about 48h with the most pronounced reduction occurring in the roots.Excised root and leaf segments from plants treated with ABAfor 24, 48 and 72 h absorbed significantly more ¹⁴C leucine,compared to the control but the percent incorporation into proteinwas not altered in roots. In response to ABA total free amino nitrogen in leaves was lowerat 5 and 72 h and in stems at 72 h. Amino nitrogen content ofroots was enhanced by ABA at 5, 24 and 72 h due to generallyhigher levels of aspartate, serine, glutamate, proline and ammonia.There was no consistent relationship between ABA suppressionof nitrate reductase activity and ammonia or specific aminoacid (except proline) levels in leaves and stems. The increasedfree amino nitrogen levels in response to the hormone may bethe result of impaired NO₃– reduction rather than thecause. The results of protein synthesis studies and solubleprotein content suggest that ABA inhibition of nitrate reductaseis not due to general inhibition of protein synthesis and mayinvolve specific inhibition of nitrate reductase protein synthesis. ¹ Contribution No. 684, Department of Plant Science, Universityof Manitoba.     

Effect of colchicine on atherosclerosis. II. Biochemical studies on skin biopsies from patients treated perorally with colchicine

The biosynthesis of proteins and glycosaminoglycans (GAGs) was determined in skin biopsies from atherosclerotic patients treated perorally for 3 months with 1 mg/day colchicine. The biopsies were incubated with 3H-glucosamine and 14C-proline for 5 h and subsequently digested with pronase. In an aliquot of the pronase digest, the specific radioactivity of 14C-proline and 14C-hydroxyproline were determined. The 3H-glucosamine-labeled GAGs were identified by specific enzymic assay and quantified after electrophoretic separation. 3 months treatment with colchicine did not modify the total amounts of proline and hydroxyproline in skin proteins, but diminished the amount of the GAGs as expressed by uronic acid content. Colchicine treatment decreased also the specific radioactivity of proline and hydroxyproline, which reflects a decrease of total protein and collagen synthesis. The incorporation of 3H-glucosamine in the 3H-GAGs was also decreased, mainly in hyaluronic acid. These results suggest that peroral administration of colchicine modifies the synthesis of extracellular matrix proteins and polysaccharides by skin fibroblasts.     

N-Acetylglucosamine Transfer Reactions and Glycoprotein Biosynthesis in Castor Bean Endosperm

N-Acetyl-[³H]glucosamine supplied to intact 3 d old castor beanendosperm tissue was incorporated into TCA-insoluble productpresumed to be glycoprotein. After an incubation time of 2 hthe major paniculate location of this product within the cellwas the endoplasmic reticulum. Cell-free preparations containingparticulate enzymes transferred N-acetyl-[¹⁴C]glucosamine fromUDP-N-acetyl-[¹⁴C]glucosamine into a fraction soluble in chloroform/methanol(2: 1, by vol), a fraction soluble in chloroform/methanol/water(10: 10: 3, by vol.), and an insoluble residue. Mild acid hydrolysisreleased the saccharide moieties from the lipids. Paper chromatographicanalysis of the released saccharides established that the C/M-solubleproducts contained both N-acetyl-[¹⁴C]glucosamine and N, N'-diacetyl-[¹⁴C]chitobiose.In contrast, N-acetyl-[¹⁴C]glucosamine released from the C/M/W-solubleproduct was contained in an oligosaccharide, probably in associationwith unlabelled mannose residues. The stimulatory effect ofdolichol monophosphate and the inhibitory effect of tunicamycinon saccharide-lipid synthesis indicated that N-acetyl-glucosamineis transferred to a glycopolymer by the established reactionsof the dolichol monophosphate pathway. The enzymes catalysingthe constituent reactions of this pathway were exclusively locatedin the ER.     

14C-Translocation in Kalanchoe pinnata at two Different Stages of Development

Mayoral, M. L. and Medina, E. 1985. ¹⁴C-translocation in Kalanchoepinnata at two different stages of development.—J. exp.Bot. 36: 1405–1413 Translocation of ¹⁴C-compounds from mature leaves was measuredin plants of Kalanchoe pinnata to determine the interactionbetween plant age and CAM phase when CO² is taken up. Matureleaves of 4 and 12 month old plants were fed with ¹⁴CO² eitherduring CAM phase 1 (midnight) or at the beginning of CAM phase4 (early afternoon). Export of ¹⁴C activity from source leaves,and distribution of ¹⁴C activity in soluble and insoluble compoundswas measured both in source leaves and sink organs. In 4 monthold plants 4 d were needed to export 76% of total ¹⁴C activityincorporated during CAM phase 1, while leaves labelled at thebeginning of CAM phase 4 exported 44% of total ¹⁴C activityafter 4 h, and 80% after 24 h. In both cases the major fractionof total radioactivity translocated was found in the roots inthe form of neutral sugars. Differences in translocation patternsare due to distribution of ¹⁴C in the source leaves, 96 % of¹⁴C taken up during CAM phase 1 is found in the insoluble fractionat the end of the subsequent phase 3, while 93 % of total radioactivitytaken up at the beginning of phase 4 is found in the solublefraction at the end of this phase. In 12 month old plants labelledduring phase 1 very little translocation could be detected atthe end of phase 3, while only 20% of total radioactivity wastranslocated from leaves labelled during phase 4 and measured4 h later. ¹⁴C activity in the older leaves had a similar distributionin soluble and insoluble fractions as the one determined inthe younger plants. Ability to translocate carbon compoundsfrom source leaves during phase 3 was shown by loading matureleaves at dawn with ¹⁴C-sucrose. Here again, mature leaves ofyounger plants showed faster translocation of radioactivitythan those of older plants Key words: Kalanchoe, crassulacean acid metabolism, translocation, sink, source relationships     

A possible role of hydroxyproline-protein in oxygen-sensitive growth

Exposure of Avena coleoptile sections to 8% O₂ brought aboutrespiration decrease, resulting in a decrease of ATP production.The pH at the cell wall surface slightly rose in sections exposedto 8% O₂, while their growth was greatly accelerated. Moreover,this growth acceleration was observed even in sections treatedwith CCCP known to make membranes permeable for protons. Weconcluded that the growth acceleration with reduction of O₂concentration is probably not the result of secretion of H₊ions into cell wall compartments. Results of this study provided evidence to support the hypothesisthat there is an inverse relationship between hydroxyproline-proteinlevel and the ability of a cell to undergo rapid cell elongation.Total labeling of the cell wall fraction with ¹⁴C-proline wasunaffected by 8% O₂ treatment, although the radioactivitiesof hydroxyproline incorporated into this fraction during thetreatments fell to about 45% of the control. Moreover, the radioactivitiesof hydroxyproline incorporated into the SLS-insoluble cell wallfraction of sections exposed to 8% O₂ decreased to about 30%of the control. This decrease of hydroxyproline was also observedin sections treated with cycloheximide, which inhibits the secretionof H₊ ions into the cell wall compartment. Reduction of O₂ concentrationin the surrounding atmosphere affects not only the hydroxylationof peptidyl proline, but also the binding of hydroxyproline-protein(s)to cell wall polysaccharides, and the resulting decrease ofthe protein rigidly bound to them may induce cell elongation. (Received December 5, 1975; )     

Utilization Modes of Inorganic Carbon for Photosynthesis in Various Species of Chlorella

Rates of CO₂ and HCC₃^– fixation in cells of various Chlorellaspecies in suspension were compared from the amounts of ¹⁴Cfixed during the 5 s after the injection of a solution containingonly ¹⁴CO₂ or H¹⁴CO₃^–. Results indicated that irrespectiveof the CO₂ concentration during growth, Chlorella vulgaris 11h and C. miniata mainly utilized CO₂, whereas C. vulgaris C-3,C. sp. K. and C. ellipsoidea took up HCO₃^– in additionto CO₂. Cells of C. pyrenoidosa that had been grown with 1.5%CO₂ (high-CO₂ cells) mainly utilized CO₂, whereas those grownwith air (low-CO₂ cells) utilized HCO₃^– in addition toCO₂. Cells that utilized HCO₃^– had carbonic anhydrase(CA) on their surfaces. The effects of Diamox and CA on the rates of CO₂ and HCO₃^–fixation are in accord with the inference that HCO₃^– wasutilized after conversion to CO₂ via the CA located on the cellsurface. CA was found in both the soluble and insoluble fractions;the CA on the cell surface was insoluble. Independent of the modes of utilization, the apparent K_m (NaHCO₃)for photosynthesis was much lower in low-CO₂ cells than in high-CO₂ones. The fact that the CA in the soluble fraction in C. vulgarisC-3 was closely correlated with the K_m(NaHCO₃) indicates thatsoluble CA lowers the K_m. ¹ Dedicated to the late Professor Joji Ashida, one of the foundersand first president of the Japanese Society of Plant Physiologists. ⁴ On leave from Research and Production Laboratory of Algology,Bulgarian Academy of Sciences, Sofia. (Received September 14, 1982; Accepted March 1, 1983)     

Metabolism of S-methylcysteine and pectin esterification in Chinese cabbage, Brassica pekinensis Rupr.

S-Methyl-L-cysteine was actively metabolized in Chinese cabbageand carbon from its methyl group was distributed into both thesoluble and insoluble fractions. The high incorporation of ¹⁴Cfrom the methyl group into the insoluble fraction after administeringof S-methyl-L-cysteine-¹⁴CH₃, and our previous results thatS-methyl-L-cysteine is demethylated to give cysteine, suggestthat S-methyl-L-cysteine might act as a methyl donor in Chinesecabbage. To obtain evidence for this possibility, incorporationof the methyl-¹⁴C of S-methyl-L-cysteine into methyl estersof pectic substances was investigated. Most of the ¹⁴C incorporatedinto pectic substances was liberated by treatment with dilutealkali and pectin esterase. The results show that S-methyl-L-cysteineacts as a methyl donor to form pectin ester. (Received October 12, 1971; )