Comparison of the effects of auxin and fusicoccin on phosphate absorption by aged potato tuber discs

Auxin (IAA, 5 × 10⁻⁵ M ) partially prevents the increase in the rate of phosphate uptake during ageing of potato tuber discs ( Solatium tuberosum L. cv. Bintje), whereas fusicoccin (FC, 10⁻⁵ M) stimulates it. After the development of enhanced phosphate transport capacity, the response to fusicoccin is greater than with fresh discs. Complementary experiments on K⁺ (⁸⁶Rb) absorption show that FC also slightly enhances the rate of K⁺ uptake, while IAA has no much effect. It is suggested that IAA acts specifically on the development of a mechanism which occurs during the ageing period, while FC action may be more directly linked to the system of phosphate transport itself.     

Influence of auxin on phosphate absorption and metabolism of phosphorylated compounds in aged potato tuber discs (Solanum tuberosum)

Ageing of potato tuber discs markedly increases the rate of phosphate uptake. This increase is partially prevented by the presence of indoleacetic acid (IAA: 50 μ M ) in the ageing medium. ³²P distribution among the various phosphorylated fractions (P₁, organic soluble phosphate, acid-insoluble phosphate) was carried out after 24 h of ageing in the presence of IAA. An equal inhibition of the labelling rates of each of the different fractions is induced by the hormone. No important effect on respiration and ATP content was observed. Moreover, IAA neither changes the total phospholipid content nor the relative distribution of ³²P between the components. These results support the hypothesis that IAA acts specifically on the development of uptake mechanisms during the ageing period.     

Wounding and the Regulation of Apoplasmic Retrieval in Source Leaf Tissue 5Spinacia oleracea L.

Kinetic studies were performed on fresh-cut and aged leaf discsof Spinacia oleracea L. in order to investigate the regulatoryprocesses involved in sugar transport across the mesophyll plasmamembrane. A comparison of the kinetic profiles for fructose,glucose, sucrose and arginine obtained on freshly-cut and ageddiscs revealed that during wound-recovery, uptake is enhanced,but that this enhancement varies considerably for the differentsubstrates tested. Variation in the saturable and first-orderkinetic components of uptake was also observed. The involvementof a phosphoinositide-signalling mechanism in the wounding processwas examined by pretreating spinach leaves with lithium. Lithiuminhibited the enhancement of uptake and this effect was reversedby the addition of miro-inositol. However, in some experimentsthe tissue appeared to be insensitive to Li⁺. Gas chromatographicanalyses performed on cut discs indicated that ethylene wasproduced in response to wounding and that the addition of cobaltto the ageing media inhibited this ethylene production. Comparativekinetic studies of control and cobalt-treated discs indicatedthat ethylene was essential for the enhancement of transportacross the plasma membrane. However, addition of ethylene touncut tissue caused only a partial increase in the uptake offructose, which indicates that some additional wound-signallingcomponent is involved. Addition of cycloheximide to the recoverymedia completely inhibited this enhancement phenomenon. Thecycloheximide-response was not due to an inhibition of ACC synthasesynthesis nor to a reduction of ATP levels. We concluded thatthe effect of cycloheximide was on protein synthesis. Our resultsare discussed in terms of possible cellular and molecular mechanismsregulating sugar transport. Key words: Sugar transport, wounding, ethylene production, spinach leaves     

Effets du GA3 et de la BAP sur la respiration,l’absorption et le métabolisme du phosphate chez des disques foliaires dePelargonium zonale après survie

Respiration rate of foliarPelargonium discs was insensitive to ageing. The addition of BAP or GA₃ to the ageing medium did not produce any effect. The presence of GA₃ or BAP in the ageing medium induced an increase (27 %) or a decrease (45 %) of the phosphate uptake. The analysis of phosphorylated compound labelling showed that these two hormones decreased³²P incorporation in the non-acid soluble fraction and increased³²P incorporation in the acid soluble organic fraction. GA₃ and BAP had little effect on the distribution of radioactivity between the different acid soluble compounds, but they increased the ATP level. These results suggest that both GA₃ and BAP increase the basal metabolism, but they seem to act differently on the development of the uptake mechanism during ageing.     

Occurrence of Localized Intense Cation Uptake Sites in the Vascular Rings of Red Beet Storage Tissue 2

Development and decline of cation uptake capacity in discs takenfrom the vascular and parenchyma rings of storage tissue ofred table beet (Beta vulgaris L.) were observed during 12 dof ageing. Uptake capacity for Na⁺ and Rb⁺ showed a steady risereaching maximums by the fourth to fifth days of ageing. Thereafter,there was a steady decline in the uptake rates. Vascular ringtissues were able to develop a greater uptake capacity for bothNa⁺ and Rb⁺ than the tissues of parenchyma rings. This difference,which was more pronounced for Rb⁺ than for Na⁺ uptake, is attributedto a combination of variations in cell density and differencesin the acquisition and retention of the cation uptake capacity.Respiration of tissue discs showed no significant rise duringageing, nor were there significant differences in the respirationof vascular and parenchyma tissues. Vascular tissues containedsignificantly more betacyanin than parenchyma tissues; and theyretained their pigment, as well as their acquired cation uptakecapacity, for a longer period during the ageing process. Key words: Cation uptake, Red beet, Vascular rings, Ageing     

Physiological Basis for a Cycloheximide-induced Soft Rot of Potatoes by Pseudomonas fluorescens

Cycloheximide renders discs of potato tissue (Solanum tuberosum,cultivar Kennebec) susceptible to soft rot by a non-pathogenicisolate of Pseudomonas fluorescens. Pectate lyases (E.C. 4.2.99.3 [EC] )are the dominant extracellular macerating agents produced bythe test organism. Potato discs aged 24 h become resistant tomaceration by purified lyase preparations. Cycloheximide blocksthe development of resistance by inhibiting suberization. Thesite of inhibition is thought to be the cycloheximide-sensitivesynthesis of phenylalanine ammonia-lyase (E.C. 4.3.1.5 [EC] ) in potatodiscs. This enzyme is necessary for production of phenolic precursorsof suberin. Comparison of tissue from a number of potato cultivarscorrelates the synthesis of phenylalanine ammonia-lyase withresistance of discs to attack by the Pseudomonad. Resistance of potato tissue to pectate lyase is also affectedby intrinsic reactions not involving suberization. Resistanceincreases in fresh unsuberized discs when tubers are transferredfrom cold storage to room temperature before use. Resistancedecreases rapidly when tubers are transferred back to the cold.The intrinsic resistance appears to increase in the surfacelayer of cells in ageing discs. It is estimated that intrinsicreactions and suberization contribute equally to resistanceof aged discs to pectate lyase maceration.     

Development of pH sensitivity of sucrose uptake during ageing of Vicia faba leaf discs

Uptake of [U-¹⁴C]-sucrose (40 m M ) by fresh and aged peeled leaf discs of broad bean ( Vicia faba L. cv. Aguadulce) has been studied. In fresh discs, uptake was nearly insensitive to external pH, whereas the pH response of absorption in discs aged for 12 h was bell-shaped, with an optimum between pH 5 and 6. At this pH, uptake was nearly twice that in fresh tissue. The passive (insensitive to carbonyl cyanide m -chlorophenylhydrazone and to cold treatment) uptake was the same in fresh or aged discs. The development of pH sensitivity of absorption did not appear when ageing was performed in the presence of 10^−H M cycloheximide or 5.7 × 10⁻⁵ M actinomycin D. Similarly, when the tissues were treated with 10⁻³ M spermidine for 2 h after excision and then aged for 10 h, the development of the pH-sensitive uptake system was inhibited. Ca²⁺ (10⁻² M ) supplied together with spermidine prevented the inhibiting effect of spermidine. The appearance of the pH-sensitive system was also markedly reduced if ageing took place in the presence of 10⁻³ M aminoethoxyvinylglycine. Autoradiographs from fresh discs and from discs aged with or without the inhibitors suggest that pH sensitivity developed more intensively in the parenchyma than in the veins.
The results suggest some caution when using excised leaf discs for studies on sucrose uptake and phloem loading. Development of pH sensitivity of uptake may require the synthesis of both DNA-dependent RNA and protein and could be related to ethylene metabolism.     

Synthesis of a Vacuolar Acid Invertase in Washed Discs of Storage Root Tissue 11Beta vulgaris L)

The synthesis of acid invertase during washing of red beet storageroots has been investigated using protein synthesis inhibitors,antibiotics, and antibodies raised against the purified invertase.Acid invertase increased during the first 3 d of washing thendecreased, with the rates of both processes dependent on temperature.Concomitantly, acid phosphatase declined throughout this period.Activity gels confirmed the increase in the level of activeinvertase protein. The increase in activity was prevented bycycloheximide, monensin, tunicamycin, and carbonyl cyanide m-chlorophenylhydrazone.Leakage of betanin was measured and ultrastructural observationsundertaken to ensure these compounds had no non-specific effects.Immunoblotting confirmed the synthesis of a new 65 kDa invertaseduring washing and its subsequent loss. The location of theinvertase was investigated by immunoblotting of proteins invacuoles isolated from fresh and washed storage root discs,and indicated that the invertase is localized in the vacuole.The results are discussed in relation to the synthesis and targetingof invertase during the changes induced by washing. Key words: Acid invertase, Beta vulgaris L., protein synthesis, protein targeting, vacuole     

The Production of Ethylene by Whole Apple Fruits and by Tissue 1

It is shown that the production of ethylene by whole apples,discs of peel prepared from them, and extracts prepared fromthe discs and supplied with linolenic acid, increases simultaneouslyduring the development of the respiration climacteric in apples.As the climacteric peak is reached, the ability of the extractsto produce ethylene declines and this is associated with a rapidloss of ethylene-producing activity on short term (up to 24h) ageing of the peel discs from which the extracts were obtained. It is suggested that the gmn.ll ethylene production by extracts(with linolenic acid) from pre-climacteric fruits, which arenot themselves evolving sufficient ethylene for its detectionin the ambient atmosphere, may be explained on the grounds thateven before the climacterio the fruit is producing small amountsof ethylene within the tissue; the tissue, as the climactericphase is approached, becomes more sensitive to ethylene andautocatalysed production of the gas then stimulates the fruitinto the respiration climacteric.     

Influence of Abscisic Acid on Phosphorus Metabolism and Respiration in Potato Tuber Discs. Comparison with the Cycloheximide Effect

Ageing of discs of potato tuber by incubation in an aerated medium, produces an increase in the rates of respiration and of phosphate uptake. The presence of cycloheximide (CHM) or abscisic acid (ABA) in the uptake medium, does not change uptake by fresh tissue over 3–4 h. On the other hand, CHM causes an inhibition of the rate of uptake by aged tissue although ABA does not. The addition of CHM or ABA to the ageing medium, prevents totally (CHM) or only partially (ABA) the increase in phosphate uptake. The analysis of ³²P-incorporation into the various phosphorylated fractions after 24 h of ageing with CHM or ABA show that CHM induces a large inhibition of the rate of uptake with an almost complete inhibition of ³²P-incorporation into the various phosphorylated fractions. By contrast, ABA produces equal inhibition of ³²P labelling of all fractions including all acid-soluble components. CHM prevents the increase in the rate of respiration, whereas ABA causes a slight stimulation. In both cases, no important effect on ATP content was observed. These results are discussed in terms of a comparison of ABA and CHM actions. They lead to the hypothesis of a specific effect of ABA on the development of the uptake mechanism.     

Changes in Lipids of Bean Seeds (Phaseolus vulgaris) and Corn Caryopses (Zea mays) Aged in Contrasting Environments

Seeds of French bean (Phaseolus vulgaris L. cv. The Prince)and caryopses of sweet corn (maize; Zea mays L.F1 hybrid Firstof All) were stored in environments of 79% relative humidityand 25 °C, 80% r.h. and 40 °C or 100% r.h. and 45 °C,giving ageing (loss of gcrminability and vigour) over periodsof months, weeks or days, respectively. The relationship betweenchanges in lipids and changes in germinability or vigour wasunaffected in general by the speed of ageing. In the corn caryopsesthere was no evidence of hydrolysis of phospholipids or peroxidationof fatty acids during ageing. In the bean seeds phosphatidicacid increased during the ageing period and phosphatyl cholinedeclined. The percentage of fatty acids as hnolenic acid initiallyfell during bean ageing, but in the slower ageing conditionsit rose again as germination reached zero. In bean, the presenceof phosphatidic acid could be a sensitive indictator of lossof vigour, but relative proportions of the different fatty acidswould be a misleading indicator of quality. Rapid artificialageing may be an adequate model, in some species, of ageingat moderate speeds and of ageing under some ambient conditions. French bean (Phaseolus vulgaris L. cv. The Prince), sweet corn (maize, Zea mays L., Fl hybrid First of All), seed ageing, phospholipids, fatty acids     

Sulfate Uptake and Respiration of Aging Potato Discs Modified by Malonic Acid and Ultraviolet Radiation

The time course of sulfate uptake in relation to respiration is shown for aging slices of potato, Solanum tuberosum L. cv. Russet. For comparative purposes, a time course for phosphate uptake is also shown. Malonic acid depressed both the respiratory rise and the sulfate accumulating ability of aging discs. A low dose of ultraviolet radiation given at the onset of aging reduced sulfate uptake by one-half without lowering respiration. We hypothesize that sulfate uptake is controlled by at least two mechanisms: one associated with oxidative metabolism and the other mediated by protein synthesis.     

Contribution à l’étude de làabsorption du saccharose par des tissus de tubercule de pomme de terre adulte (Solatium tuberosum L.) modalités et influence des phytohormones

Sugar uptake by potato tuber discs was studied. Discs were used “fresh” or after a 24-h ageing period. It was shown that ageing increases (by 3 to 4 times) the rate of glucose and sucrose uptake. Sucrose uptake by fresh tissues was insensitive to the presence of glucose or fructose while a competitive effect was observed after ageing. This indicates the development of an invertase activity, which was inhibited by tris-buffer. Sucrose and glucose uptake by aged discs was dependent on cellular metabolism as shown by the sensitivity to low temperature and metabolic inhibitors (NaCN, DNP, CCCP). Involvement of thiol groups was demonstrated by the inhibition with NEM and PCMBS. Orthovanadate, which decreases phosphate uptake by 85 % (Poderet al. 1986) did not produce any effect on glucose and sucrose uptake by aged tissues. Fusicoccin produced only a slight stimulation (15 %). These results argue in favour of the involvement of specific ATPases in ion and sugar uptake. No involvement of a redox system was observed. ABA and BAP inhibited the uptake induced by ageing but had no effect on the endogenous sugar content. BAP would act by its effect on the amount of ATP while ABA would act at the membrane level. The results are discussed in relation to the mechanisms of transfer of glucosyl groups and to the transport of sucrose by the symplasmic pathway. Reçule     

The Organic Acid Metabolism of Swede 'Root' Tissue

Data are recorded on the quantitative changes in the contentof succinic, malic and citric acids of discs of washed ‘root’tissue of ‘swede’ (Brassica napus), subjected toexperimental treatments involving the addition of succinate,malate, citrate, -ketoglutarate, pyruvate and malonate. Theresults are interpreted as showing the acids to be associated,in part at least, with the operation of an active Krebs cycle.It is suggested that they are also involved, by providing carbonskeleton material, in the enhanced protein synthesis resultingon washing discs of swede tissue and treating washed discs withphosphate buffer.     

Regulation of development of leucine uptake activity by glutamine in the scutellum of germinating barley grain

Scutella from ungerminated grains of barley (Hordeum vulgare L. cv Pirkka) take up leucine at a slow rate, which increases rapidly during germination. When endosperms were removed from the grains after imbibition for 4 hours or after germination for 12 or 72 hours, the increase in the rate of leucine uptake was greatly accelerated during subsequent incubation of the embryos or scutella. These increases were rapidly inhibited by cordycepin and cycloheximide, suggesting that protein synthesis, probably synthesis of the carrier protein, was required for the development of the uptake activity.

In separated embryos or scutella, the increases in the leucine uptake activity were inhibited by glutamine. The inhibitions caused by glutamine and cycloheximide were not additive, suggesting that glutamine did not interfere with the function of the carrier but repressed its synthesis. Glutamine did not inhibit the simultaneous increase in peptide uptake; in this respect, its effect was specific for leucine uptake, which appears to be due to a general amino acid uptake system.

Some other protein amino acids also inhibited the increase in leucine uptake without inhibiting the increase in peptide uptake. However, these effects were smaller than that of glutamine.

These results suggest that the transfer of leucine (and other amino acids) from the endosperm to the seedling in a germinating barley grain is regulated at the uptake step by repression of the synthesis of the amino acid carrier protein by glutamine and—possibly to a lesser extent—by some other amino acids taken up from the endosperm.

     

The effects of tobacco mosaic virus synthesis on the free amino acid and amide composition of the host

1. Changes in concentrations of free amino acids and amides have been determined in TMV-infected tobacco leaf discs and in comparable uninfected discs during the time of virus formation. 2. During the period of rapid virus formation the infected discs show a transitory deficiency (as compared to uninfected discs) in glutamine, asparagine, aspartic acid, glutamic acid, serine, and to a lesser extent in valine, threonine, and proline. About 100 hours before this time smaller deficiencies in the concentrations of these components also occur. The latter effect is probably associated with the early synthesis of a non-virus protein in infected tissue. 3. Comparison of the above effects with the known amino acid composition of TMV indicates that it is unlikely that the virus protein is synthesized by condensation of appropriate free amino acids. Rather, the deficiencies observed appear to result from removal of ammonia from the nitrogen pool during synthesis of new proteins in infected tissue. Equilibrium shifts resulting from ammonia withdrawal probably account for the observed deficiencies in amides and free amino acids. TMV protein, therefore, appears to be synthesized de novo, from non-protein nitrogen, probably ammonia. 4. It is suggested that the changes in free amino acid concentrations induced by virus formation may account for some of the symptoms observed in infected plants.     

Preferential Accumulation by Mesophyll Cells at Low and by Veins at High Exogenous Amino Acid and Sugar Concentrations in Commelina benghalensis L. Leaves

The contribution to solute uptake by mesophyll cells and veinsin leaf discs, was assessed through a study of uptake in relationto concentration for ¹⁴C-labelled substrates (sucrose, glucose,arginine, proline, valine and -aminoisobutyric acid) using isolatedmesophyll cells and stripped leaf discs of Commelina benghalensisL. Uptake per unit fresh weight was higher in mesophyll cellsthan in discs at low substrate concentrations (lower than about0·5 mol m^–3). At higher concentrations, uptakeby discs exceeded that by mesophyll cells except for glucoseuptake which was higher in mesophyll cells over the whole concentrationrange. The profiles of uptake versus concentration displayedbiphasic kinetics in mesophyll cells and discs. Comparison ofthe uptake characteristics obtained by iterative fitting confirmedthat the high-affinity systems of uptake prevail in the mesophyllcells, whereas the low-affinity systems are dominant in theveins. The results provide good evidence that, supplementaryto direct vein loading, a pathway via the mesophyll contributesstrongly to the photosynthate loading by veins in stripped discs. Key words: Commelina benghalensis L., amino acid uptake, mesophyll, minor veins, phloem loading, sugar uptake     

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     

Characteristics of Sugar, Amino Acid and Phosphate Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing seeds of Vicia fabaL. and Pisum sativum L., the ‘empty’ ovules werefilled with a standard solution (pH 5.5). Seed coat exudatesof both species were collected during relatively long experiments(up to about 12 h) and the concentration of sugar (mainly sucrose),amino acids and phosphate in the exudate measured. A discussionis presented on the amino acid/sugar ratio and the phosphate/sugarratio in the seed coat exudate. A pretreatment (15 min) withp-chloromercuribenzenesulphonic acid (PCMBS) reduced the releaseof sugar, amino acids and phosphate from broad bean seed coats.After excision of ‘empty’ ovules of Vicia faba andPisum sativum from the maternal plant, 2–4 h after thistreatment a strong difference became visible between sucroserelease from excised seed coats and sucrose release from attachedseed coats. Similarly, when the rate of phloem transport ofsucrose into an ‘empty’ ovule of Vicia faba or Pisumsativum was reduced by a sub-optimal mannitol concentrationin the solution, a reduced rate of sugar release from the seedcoat could be observed. Excision and treatment with a sub-optimalmannitol concentration reduced the release of amino acids toa lesser extent than for sucrose. These treatments did not reducethe rate of phosphate release from the seed coat. Key words: Seed development, Seed coat exudate, Phloem transport