Relationship between photosynthetic carbon metabolism and essential oil biogenesis in peppermint under Mn-stress

Changes in growth and yield parameters, and ¹⁴CO₂ and (U-¹⁴C)sucrose incorporation into the primary metabolic pool, and essentialoil have been investigated under Mn-deficiency and subsequentrecovery in Mentha piperita, grown in solution culture. UnderMn-deficiency, CO₂ exchange rate, total chlorophyll, total assimilatoryarea, plant dry weight, and essential oil yield were significantlyreduced, whereas chlorophyll a/b ratio, leaf area ratio andleaf stem ratio significantly increased. In leaves of Mn-deficientplants, ¹⁴CO₂ incorporation into the primary metabolic pool(ethanol-soluble and -insoluble) and essential oil were significantlylower, whereas (U-¹⁴C) sucrose incorporation into these componentswas significantly higher as compared to the control. Among theprimary metabolites, the label was maximum in sugars, followedby organic acids and amino acids. A higher label in these metaboliteswas, in general, observed in stems of Mn-deficient plants ascompared to the control. Mn-deficient plants supplied with completenutrient medium for 3 weeks exhibited partial recovery in growthand yield parameters, and essential oil biogenesis. Thus, underMn-deficiency and subsequent recovery, the levels of primaryphotosynthetic metabolites and their partitioning between leafand stem significantly influence essential oil biogenesis. Key words: Mentha piperita, Mn-stress, ¹⁴CO₂ and [U-¹⁴C] sucrose incorporation, oil accumulation, primary photosynthetic metabolites     

Metabolism of Exogenous Malonate by Coffee Leaf Tissue

When [l-¹⁴C]-malonate was supplied to discs cut from matureleaves of Coffea arabica, ¹⁴CO₂ was released (approximately12% of the total CO₂ respired) and organic acids of the Krebscycle, uronic acids, sugars and amino acids became radioactive.There was no incorporation of MC into either lipids or phenoliccompounds. The formation of glucose from malonate has not beenobserved in other studies with plant tissues. The synthesisof labelled glucose together with an active pentose phosphatepathway that is stimulated by malonate explains the accumulationof radioactive phosphogluconate in the leaf discs. Tentativeproposals are made for pathways to account for the results obtained. Key words: Coffee leaves, Malonate metabolism, Pentose phosphate pathway     

Mobilization of Reserves and Synthesis of Sterols and Triterpenes in Seedlings of Two Canarian Euphorbia Species

Seedlings from Euphorbia canariensis and Euphorbia lambii weregrown in the dark at 25 °C. Protein and triglyceride contentas well as levels of sugars and amino acids in the endospermwere determined during endosperm depletion. In the endospermof Euphorbia canariensis, relatively low levels of amino acids(up to 1 µmol.endosperm^–1) were found of which glutamine/glutamateaccounted for 40% at the stage of radicle emergence. High levelsof amino acids (up to 4 µmol.endosperm^–1) comparedwith sugars (up to 2 µmol sucrose.endosperm^–1) weredetected in the endosperm of Euphorbia lambii. Arginine wasthe main component (28 µmol%) of the amino acids in thistissue. In both species amino acid composition changed graduallyduring endosperm depletion. Cotyledons retained their ability to absorb a variety of watersoluble substrates after removal of the endosperm. ¹⁴C from[U-¹⁴C]sucrose was effectively incorporated into the triterpenesof the laticifers and to a lesser extent into the sterols ofthe seedling. The highest incorporation values were found inyoung seedlings about 2 d after the emergence of the radicle.Seedlings of this age also showed high incorporation rates of¹⁴C from labelled alanine, serine, threonine, valine, leucineand isoleucine into both triterpenols and sterols, but no generalconclusions about metabolic channelling in lipid synthesis couldbe made. Endosperm, Euphorbia canariensis L. Euphorbia lambii Svent., sterols, triterpenols, amino acids, laticifer, biosynthesis     

Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport in Cucurbita pepo L. Hypocotyl Segments

Pretreatment of 2?0 mm segments of etiolated zucchini (Cucurbitapepo L.) hypocotyl with cycloheximide (CH) or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide(MDMP) eliminated the stimulation by N-1-naphthylphthalamicacid (NPA) of net uptake of [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA),but had relatively little effect on the net uptake of IAA inthe absence of NPA. The efflux of [1-¹⁴C]IAA from preloadedsegments was not substantially affected by inhibitor pretreatmentin the absence of NPA, but CH pretreatment significantly inhibitedthe reduction of efflux caused by NPA. Pretreatment with CHor MDMP did not affect net uptake by segments of the pH probe[2-¹⁴C]5,5-dimethyl-oxazolidine-2,4-dione ([2-¹⁴C]DMO), or thenet uptake of [¹⁴C]-labelled 3-O-methylglucose ([¹⁴C]3-0-MeGlu),suggesting that neither inhibitor affected intracellular pHor the general function of proton symporters in the plasma membrane.Both compounds reduced the incorporation of label from [³⁵S]methionineinto trichloroacetic acid (TCA)-insoluble fractions of zucchinitissue, confirming their inhibitory effect on protein synthesis. The steady-state association of [³H]IAA with microsomal vesiclesprepared from zucchini hypocotyl tissue was enhanced by theinclusion of NPA in the uptake medium. The stimulation by NPAof [³H]IAA association with microsomes was substantially reducedwhen the tissue was pretreated with CH. However, CH pretreatmentdid not affect the level of high affinity NPA binding to themembranes indicating that treatments did not result in lossof NPA receptors. It is suggested that the auxin transport site on the effluxcarrier system and the receptor site for NPA may reside on separateproteins linked by a third, rapidly turned-over, transducingprotein. Key words: Auxin carriers, auxin efflux, Cucurbita pepo, phytotropin receptors     

Germination of Phaseolus vulgaris III. The role of nucleic acid and protein synthesis in the initiation of axis elongation

Excised embryonic axes of Phaseolus vulgaris L. (var. WhiteMarrowfat) begin cell elongation after approximately 4 hr ofincubation at 26°C. The incorporation of ³²P into nucleicacids and phenylalanine-l-¹⁴C into protein markedly increasesduring the 4th hr of incubation, prior to initiation of cellelongation. CH, which inhibits incorporation of phenylalanine-l-¹⁴C intoprotein by 93% during the 2nd hr after its addition, completelyprevents the initiation of axis elongation if added up to 2hr after the beginning of imbibition. Actinomycin D reducesthe fresh weight increase of the axes, and inhibits both ³²Pincorporation into nucleic acids and phenylalanine-l-¹⁴C incorporationinto protein. 5-FU inhibits ³²P incorporation into nucleic acidsbut not phenylalanine-l-¹⁴C incorporation into protein or thefresh weight increase of the axes. MAK column chromatography indicates that actinomycin D inhibitsthe synthesis of all types of nucleic acids to about the sameextent, while 5-FU almost completely inhibits the accumulationof ³²P in ribosomal RNA with lesser but significant inhibitoryeffects on accumulation of ³²P in tRNA. The results suggest an absolute requirement for protein synthesisprior to initiation of cell elongation and at least a partialrequirement for synthesis of nucleic acid species other thanribosomal RNA, tRNA and DNA. The kinetic data suggest that theaxes develop a greatly increased capacity for nucleic acid andprotein synthesis prior to initiation of axis elongation. ¹This research was supported by NSF grant GB 4145 and a grantfrom the U. S. Forest Service. (Received December 16, 1968; )     

Intracellular amino acids and protein synthesis in Hela cells

1. When the intracellular amino acid pool is prelabelled and subsequently chased in non-radioactive medium, the radioactivity of the amino acid pool is not found to have been incorporated into protein.
2. Leucine transport into Hela cells is reduced in the presence of 10 mM valine in the medium. This results in a lower specific radioactivity of leucine in the intracellular amino acid pool. However, neither the overall rate of protein synthesis nor the incorporation of radioactive leucine into protein is affected.

From these experiments it is concluded that incoming amino acids entering the intracellular amino acid pool are not used for de novo synthesis of protein.     

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.     

Light rather than iron controls photosynthate production and allocation in Southern Ocean phytoplankton populations during austral autumn

The role of iron and light in controlling photosynthate productionand allocation in phytoplankton populations of the Atlanticsector of the Southern Ocean was investigated in April–May1999. The ¹⁴C incorporation into five biochemical pools (glucan,amino acids, proteins, lipids and polysaccharides) was measuredduring iron/light perturbation experiments. The diurnal Chla-specific rates of carbon incorporation into these pools didnot change in response to iron addition, yet were decreasedat 20 µmol photons m^–2 s^–1, an irradiancecomparable with the one at 20–45 m in situ depth. Thissuggests that the low phytoplankton biomass encountered (0.1–0.6µg Chl a L^–1) was mainly caused by light limitationin the deep wind mixed layer (>40 m). Regional differencesin Chl a-specific carbon incorporation rates were not foundin spite of differences in phytoplankton species composition:at the Antarctic Polar Front, biomass was dominated by a diatompopulation of Fragilariopsis kerguelensis, whereas smaller cells,including chrysophytes, were relatively more abundant in theAntarctic Circumpolar Current beyond the influence of frontalsystems. Because mixing was often in excess of 100 m in thelatter region, diatom cells may have been unable to fulfil theircharacteristically high Fe demand at low average light conditions,and thus became co-limited by both resources. Using a modelthat describes the ¹⁴C incorporation, the consistency was shownbetween the dynamics in the glucan pool in the field experimentsand in laboratory experiments with an Antarctic diatom, Chaetocerosbrevis. The glucan respiration rate was almost twice as highduring the dark phase as during the light phase, which is consistentwith the role of glucan as a reserve supplying energy and carbonskeletons for continued protein synthesis during the night.     

Germination of wheat embryos and the transport of amino acids into a protein synthesis precursor pool

Wheat (Triticum aestivum L. var. Lew) embryonic axes take up externally supplied radioactive amino acid (from a solution greater than 2 millimolar) such that the specific radioactivity of the total internal amino acid rapidly reaches that of the external solution. Nevertheless, incorporation of radioactive amino acid into protein increases steadily as the concentration of external amino acid is increased, indicating that the amino acid that is precursor to protein synthesis is not in equilibrium with the total internal amino acid pool. When the external source of amino acid is removed, incorporation of radiolabeled amino acid into protein continues at a rate comparable to that of embryos maintained in the radioactive solution. In explanation of these data, it is suggested that there are two separate cytoplasmic pools of amino acids, one a protein synthesis precursor pool, and the second, an expandable pool into which exogenous radioactive amino acids are taken up. The protein synthesis pool is fed at a limited rate from the expandable pool and at a far greater rate from an endogenous source. As a consequence, the specific activity of the amino acid that is the precursor for protein synthesis is considerably below that of the total internal pool and is determined by the rate of movement into the protein synthesis pool from the expanded radioactive cytoplasmic pool.

The rate of movement of amino acids from the expandable pool into the protein synthesis pool increases approximately 5-fold during the initial 4.5 hours of embryo germination. When this change is considered in analyzing the relative rates of protein synthesis, there is probably no more than a 2-fold increase in protein synthetic capacity between embryos germinated for 1.5 and 4.5 hours. The leveling off of the change in transport capacity after 4.5 hours suggests that the earlier increase in the rate of this process may be a necessary step before the embryos can begin to accelerate their growth rate.

     

The Partitioning of Photosynthetically Assimilated 14C into Amino Acids in Wheat: 2. Distribution in Amino Acids of Wheat Grain Fractions

Changes in the distribution of ¹⁴C between free and bound aminoacids in wheat grains (Triticum aestivum L. cv. Arkas) at 10and 20 d post-anthesis are described. After ¹⁴CO₂, labellingof the flag leaf, ¹⁴C was initially more rapidly transferredto the grains of 20 d post-anthesis plants than for 10 d post-anthesisplants. However, after a 460 min chase period in the light theamount of ¹⁴C in the grains of the younger and older plantswere similar. In the younger, more rapidly growing grains, agreater proportion of the ¹⁴C was incorporated into structuraltissue and starch. ¹⁴C accumulation in the grains continuedduring the dark in the younger grains but not in the older grains. Although the overall ¹⁴C distribution between the free aminoacid and protein pools of the grain was similar for both treatments,the distribution within the albumin, prolamin and globulin fractionsand between the individual non-bound amino acids differed. Ofthe protein fractions, the albumins were initially the mostheavily labelled but after 460 min chase the prolamins containedmore ¹⁴C. The majority of the ¹⁴C in the albumin and globulinfractions after 280 min chase was in hydrolysable, non-aminoacid compounds. In both tissues, the free amino acid pools lostradioactivity in the dark but the solid residues and proteinscontinued to function as ¹⁴C sinks. Daily fluctuations in the radioactivity in free and bound alanineare consistent with the role of free alanine as a diurnal metabolicnitrogen pool. Wheat, Triticum aestivum¹⁴CO₂, amino acids, proteins, carbon metabolism     

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; )     

Metabolism of Inorganic Carbon Taken Up by Roots in Salix Plants

The metabolic products of inorganic carbon taken up throughthe roots from nutrient solution were studied in willow plants.Willow cuttings (Salix cv. Aquatica gigantea) were suppliedwith unlabelled or ¹⁴C-labelled NaHC0₃ for 1, 5, 10, and 24h in light or in darkness. After feeding, the plants were dividedinto six samples (upper and lower leaves and corresponding stems,cuttings and roots), which were frozen in liquid N₂. Freeze-driedground samples were extracted into water-soluble, chloroform-solubleand insoluble fractions. The water-soluble fraction was furtherseparated into basic, acidic, and neutral fractions by ion-exchangechromatography. In the light experiment pronase treatment wasused to separate the insoluble fraction into proteins and insolublecarbohydrates. After I h feeding time, most of the ¹⁴C was fixed into organicacids and amino acids both in light and in darkness in all partsof the plants. In the roots a large part of the ^l4C-carbon wasincorporated into the protein and insoluble fractions alreadyduring short feeding times, and the amounts incorporated increasedwith time. In the leaves, after 1 and 5 h the main labelledcompounds were the organic acids and amino acids, but after10 h about half of the total ¹⁴C was in protein and in the insolublefraction. A further analysis of amino acids and organic acidswith HPLC showed that C-4 acids were labelled initially andthat over time the proportion of different acids changed. These results indicate that the metabolism of carbon in rootsmight take place via ß-carboxylation of PEP. Partof the fixed ¹⁴C is transported from the roots, probably asamino acids and organic acids, to the shoot. In roots the C-4acids are metabolized further into structural compounds (proteinsand insoluble carbohydrates). Key words: DIC, Salix, roots, metabolism, HPLC     

Origin of Amino Acids in Datura stramonium Seeds

¹⁴C isotope studies show that the seeds of Datura stramoniumL. can produce a number of amino acids (particularly alanine,glutamate, phenylalanine, and tyrosine) from a supply of sucroseand nitrate. These amino acids can be incorporated into theseed protein. The bulk of the amino acids incorporated into the seed proteinmust, however, be supplied by adult leaves in the proximityof the fruit, either as the amino acids themselves, or theirimmediate precursors. The major free-amino-acid products of Datura leaves are theamides asparagine and glutamine.     

ORIGIN OF AMINO ACIDS CONSTITUTING CELLULAR PROTEIN IN GERMINATING CONIDIOSPORES OF ASPERGILLUS NIGER

Formation of pool amino acids in germinating spores of Aspergillusniger strain 1617 was investigated. The pool amino acids comprisedmainly glutamic acid and alanine. Small amounts of pyruvateand -ketoglutarate were found to increase almost in parallelwith the course of increase in the amount of free amino acidsup to the stage of onset of active protein synthesis. Asparticglutamictransaminase activity was exhibited even in dormant spores andit developed in response to the increase in cellular protein.Alanine-glutamic transaminase activity, on the other hand, waslacking in dormant spores and appeared at the stage of accumulationof amino acids preceding protein synthesis. It was revealed from the experiments with ³⁵S-labeled sporesthat the dormant spores of this fungus contain two unidentifiedsulfur substances, and the sulfur of these substances is incorporatedinto the sulfur amino acids of the protein synthesized in germinatingspores. ¹Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo (Received September 11, 1959; )     

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 Partitioning of Photosynthetically Assimilated 14C into Amino Acids in Wheat 1. Partitioning During Transport to the Grain

When ¹⁴CO₂ was fed to flag leaf laminae at 20 d post-anthesis,the transport organs between the leaf and the grains containedappreciable ¹⁴C in glutamine, glutamate, serine, alanine, threonineand glycine. Smaller amounts of ¹⁴C were present in gamma-aminobutyricacid (GABA), aspartate and cysteine. Other amino acids whichwere labelled in the source leaf were not labelled in the transportorgans. The export of labelled glutamine, serine, glycine andthreonine from the source leaf was favoured in comparison tothe other amino acids mentioned. Threonine accumulated, andwas subsequently metabolised, in the rachis. [¹⁴C]GABA alsoaccumulated in the rachis. In the grains, the relative amountof soluble [¹⁴C]alanine increased with chase time. This wasprobably due to de novo synthesis and reflected the specialrole of alanine in grain nitrogen metabolism. Wheat, Triticum aestivum, ¹⁴CO₂, amino acids, transport, carbon metabolism     

Studies on soluble RNA binding indoleacetic acid in etiolated mung bean hypocotyl sections

Nucleic acid was extracted by the SLS-phenol method from Phaseolusaureus hypocotyl treated with IAA-2-¹⁴C. Radioactivity in thenucleic acid fraction was found at the positions of sRNA andrRNA on an MAK column. IAA-¹⁴C was released from the radioactivecompound(s) in the sRNA fraction, by alkaline hydrolysis, butnot by ethanol extraction, or by dialysis to 2 M NaCl, 8 M urea,and 0.1 M EDTA. When the radioactive compound at the positionof sRNA on an MAK column was further re-chromatographed on aDEAE-cellulose column and on a BD-cellulose column, it was alwayslocalized only in a settled part of the fraction of each column.From this fraction IAA-¹⁴C was released by alkaline hydrolysis.Also, IAA-¹⁴C was released from the radioactive compound insRNA fraction, by RNase digestion, but not by pronase treatment.Results of these experiments suggest the existence of some kindsof sRNA binding IAA. The genesis of this sRNA binding IAA-¹⁴Cwas observed within 30 min after the supply of IAA-¹⁴C, andthe sRNA became saturated with IAA-¹⁴C about 2 hr after thebeginning of incubation. The behavior of sRNA binding IAA, representedby sRNA binding IAA-¹⁴C, may have a role in IAA induced growthof mung bean hypocotyl sections. (Received July 6, 1971; )     

[14C]-Glucose Metabolism during Shoot Bud Development in Cultured Cotyledon Explants of Pinus radiata

Excised cotyledons of Pinus radiata D. Don cultured under shoot-forming(plus benzyladenine) and non shoot-forming (minus benzyladenine)conditions for 10 and 21 days were fed U-[¹⁴C]-glucose for 3h in the light followed by a 3 h chase period. The labellingof individual metabolites as well as ¹⁴C incorporation intoprotein was assessed. It was found that the general metabolicpatterns were qualitatively the same in shoot-forming and nonshoot-forming conditions, however, metabolism leading to respirationas well as to the synthesis of some amino acids and proteinsynthesis was enhanced in the shoot-forming cultures. (Received February 16, 1987; Accepted July 8, 1987)     

Total and Polar Lipid Biosynthesis during Crotalaria juncea Linn. Pollen Tube Growth: Effect of Gibberellic Acid, Indoleacetic Acid and (2-Chloroethyl)-phosphonic Acid

Gibberellic acid (GA₃) at 58 µM, indoleacetic acid (IAA)at 29 µM, and (2-chloroethyl) phosphonic acid (Ethephon)at 70 µM promoted pollen tube growth in Crotalaria junceapollen suspension cultures both in water and basal medium. GA₃stimulated [ l-¹⁴C]acetate incorporation into total lipids inboth media, whereas IAA enhanced incorporation in water culturesonly. On the contrary, Ethephon reduced the label in total lipidswhen supplemented in basal medium. Based on [l-¹⁴C lacetateincorporation into different phospho- and glycolipids, it isproposed that these growth regulators have a definite role inthe biosynthesis of lipid components of the membranes.     

Heat Shock Proteins of Cyanobacterium Anacystis nidulans

Cells of Anacystis nidulans grown at 30°C were incubatedwith ¹⁴C-Chlorella protein hydrolysate at the elevated temperatures(30–55°C) and the effect of heat shock treatment onprotein synthesis was studied. Incubation temperatures higherthan 45°C caused a significant decrease in the incorporationof amino acids into proteins. Further, the heat shock treatmentinduced significant changes in the fluorographic profile ofthe newly synthesized proteins. (Received October 25, 1985; Accepted December 4, 1985)