The Effects of Aqueous Extracts of Red Beet Root on Salt Accumulation and Respiration of Discs of Red Beet Root

An account is given of the preparation of aqueous extracts ofred beet root which are shown to stimulate potassium uptakein beet discs washed for a short period, but inhibit potassiumuptake in discs washed for four days or more. Analysis of extractsshowed them to contain organic anions (especially citrate andmalate) which affect both the metabolic phase of potassium uptakeand respiration of the tissue. The effects of extracts and organicacids on uptake of manganese by beet discs is described andcompared with effects on potassium absorption. The results arediscussed with respect to current theories of salt accumulationand in relation to the hypothesis relating an inhibitor of saltaccumulation to the lag phase of ion uptake by beet discs.     

Kinetics of uptake and release of Cd by potato tissue

Abstract. The absorption of Cd by potato tissue was studied. No enhancement of the absorption capacity for Cd was observed by storage of the sliced potato tissue in aerated doubly distilled water for a period of 48 h. Cd absorption as a function of the external Cd concentration followed a typical hyperbolic isotherm. A two-phase uptake process for the absorption of Cd was found: a rapid initial phase terminating after 30 min, was followed by a slower phase with a constant rate. Efflux, studied by means of compartmental analysis, of Cd showed three phases. The two first were shown to be extracellular in origin. The third phase, involving Cd remaining in the discs after efflux, probably involved Cd in different forms. Metabolic processes, however, did not seem to contribute to this phase.     

Multiphasic absorption of glucose and 3-o-methyl glucose by aged potato slices

The isotherm for glucose absorption by aged potato (Solanum tuberosum var. Russet Burbank) discs shows four distinct phases in the concentration ranges 1.0 to 75 μm, 75 μm to 1.5 mm, 1.5 to 15 mm, and 15 to 100 mm, respectively. Each segment of the multiphasic isotherm, when plotted reciprocally by the method of Lineweaver and Burk or of Hofstee, without regard for uptake in earlier phases, indicates absorption rate to be a hyperbolic function of concentration. The observations suggest that glucose uptake is carrier-mediated, and that the transport barrier undergoes a series of all-or-none transformations at critical external concentrations, yielding successive new and higher values for the parameters Km and V_max 3-O-Methyl glucose, a nonmetabolizable analogue of glucose, shows the same multiphasic absorption isotherm, with Km values essentially similar to those for glucose uptake, and V_max values somewhat lower than those for glucose absorption. Whereas the first three phases of the absorption isotherm are taken to reflect passage across the plasma membrane, the fourth phase may reflect kinetics of glucose or 3-O-methyl glucose transport to the vacuole.     

Physiological Loading Can Restore the Proteoglycan Content in a Model of Early IVD Degeneration

A hallmark of early IVD degeneration is a decrease in proteoglycan content. Progression will eventually lead to matrix degradation, a decrease in weight bearing capacity and loss of disc height. In the final stages of IVD degradation, fissures appear in the annular ring allowing extrusion of the NP. It is crucial to understand the interplay between mechanobiology, disc composition and metabolism to be able to provide exercise recommendations to patients with early signs of disc degeneration. This study evaluates the effect of physiological loading compared to no loading on matrix homeostasis in bovine discs with induced degeneration. Bovine discs with trypsin-induced degeneration were cultured for 14 days in a bioreactor under dynamic loading with maintained metabolic activity. Chondroadherin abundance and structure was used to confirm that a functional matrix was preserved in the chosen loading environment. No change was observed in chondroadherin integrity and a non-significant increase in abundance was detected in trypsin-treated loaded discs compared to unloaded discs. The proteoglycan concentration in loaded trypsin-treated discs was significantly higher than in unloaded disc and the newly synthesised proteoglycans were of the same size range as those found in control samples. The proteoglycan showed an even distribution throughout the NP region, similar to that of control discs. Significantly more newly synthesised type II collagen was detected in trypsin-treated loaded discs compared to unloaded discs, demonstrating that physiological load not only stimulates aggrecan production, but also that of type II collagen. Taken together, this study shows that dynamic physiological load has the ability to repair the extracellular matrix depletion typical of early disc degeneration.     

Modulation by weak bases or weak acids of the pH of cell sap and phosphoenolpyruvate carboxylase activity in leaf discs of C4 plants

When leaf discs of a C₄ species, Alternanthera pungens (L.) H.B. and K. or Amaranthus hypochondriacus L., were preincubated in 7.5 m M NH₄Cl, the pH of the cell sap increased by nearly 0.3 unit, while the activity of phosphoenolpyruvate carboxylase (PEPC) about doubled compared to the cell sap from control leaf discs (preincubated in 5 m M Tricine‐KOH, pH 8.5). The sensitivity of PEPC to L ‐malate (a feedback inhibitor) decreased marginally as a result of cytosolic alkalization. The pH of the cell sap and PEPC activity decreased by nearly 0.4 unit and 50%, respectively, when leaf discs were incubated in weak organic acids such as propionic, butyric or salicylic acid. Thus, our results demonstrate a marked modulation in vivo of cell sap pH and PEPC activity in leaf discs from C₄ plants by external alkalizing or acidifying reagents. The rise in PEPC activity due to alkalization of leaf discs was not sensitive to cycloheximide, implying that cytosolic protein synthesis was not involved in the activation of PEPC. Despite the marked increase in the PEPC activity due to the base‐loading of leaf discs, the change in malate sensitivity of the enzyme was only marginal, indicating that there was no significant increase in the extent of PEPC‐phosphorylation. Besides the physiological significance, the technique of acid/ base‐loading may be an important tool for studying the regulation of PEPC in leaf discs of C₄ species, since the activity of PEPC could be enhanced apparently without phosphorylation of the enzyme.     

The Absorption and Accumulation of Salts by Living Plant Cells: IX The Absorption of Rubidium bromide by Potato Discs

Chemical methods for the quantitative determination of rubidiumin aqueous plant extracts and saps were investigated but abandonedin favour of a spectrographic technique which is fully described. The results of quantitative determinations of the rubidium andbromide absorbed by potato discs from solutions of rubidiumbromide, and the effects upon this absorption of oxygen, oftime, and of the specific surface of the discs are described.The effects of these variables upon the accumulation of bromidewhich were previously described have been confirmed. All thevariables which affect bromide absorption affect the uptakeof rubidium in a similar manner. The absorption of rubidium is of two distinct kinds. The firstphase in the absorption process is a relatively rapid uptakeof rubidium unaccompanied by bromide. This process is unaffectedby oxygen, is not confined to the surface cells, and ceasesafter a short time. The second phase occupies a protracted periodduring which both rubidium and bromide are absorbed in equivalentamounts. During this phase the absorption is confined to a fewlayers of cells at the surface and is determined by oxygen concentrationin the manner already described for the bromide ion. The two types of absorption are described as ‘inducedabsorption’ and as ‘primary absorption’ inthe sense used earlier. The former is merely a property of thesubstances in the tissue, the latter is a process which tendsto increase the free energy of the tissue, therefore demandsthat work should be done, and is a property of the organizedliving cell. The relationships of the two types of absorption process totime have been described and they can be expressed in termsof equations. The effect of the surface and thickness of discs upon the absorptionof bromide and rubidium is interpreted quantitatively. The distance from the surface of the discs at which ‘primaryabsorption’ ceases is the same for rubidium and bromide,and coincides with the values previously determined for thedepth of the layer of tissue which exhibits enhanced respiration. The ‘induced absorption’ of rubidium may be observedin discs killed by alcohol. The effect of concentration uponthis process is similar to the adsorption isotherm. The effectof concentration upon the absorption of bromide by living discsis much less conspicuous and the requirements of the absorptionisotherm are not rigidly met. The effect of concentration alsoinvolves the factor of surface and thickness of disc. Unequal absorption of the bromide and rubidium arises from thesuperimposed effect of two distinct processes one of which causesthe absorption of rubidium only and the other tends to causeequal uptake of rubidium and bromide.     

Dihydroflavonol Reductase Activity in Relation to Differential Anthocyanin Accumulation in Juvenile and Mature Phase Hedera helix L

Juvenile phase English ivy (Hedera helix L.) plants accumulate anthocyanin pigment in the hypodermis of stems and petioles, whereas genetically identical plants of the mature phase do not. The objective of this work was to assess which enzyme(s) might limit anthocyanin accumulation in mature phase ivy. Leaf discs of both juvenile and mature phase ivy accumulated comparable levels of the flavonols kaempferol and quercetin, whereas only juvenile phase discs accumulated anthocyanin. The accumulation of quercetin, but lack of accumulation of leucocyanidin or anthocyanin in mature phase discs, suggested that mature discs lacked dihydroflavonol reductase activity. There was no detectable dihydroflavonol reductase activity in mature phase discs, whereas there was an induction of activity in juvenile phase discs in response to sucrose, or photosynthetically fixed carbon, and light as a photomorphogenic signal. Phenylalanine ammonia-lyase, an enzyme early in the anthocyanin biosynthetic pathway, was induced above its basal level by sucrose and light in discs of both phases of ivy, with greater activity in mature phase discs. Phenylpropanoids, a class of compounds that are precursors to flavonoids, accumulated in leaf discs of both phases, with greater levels in mature phase discs. These results indicate that the lack of dihydroflavonol reductase activity limits the accumulation of anthocyanin in mature phase tissue.     

Adenosine nucleotides and ATP synthesizing enzymes in conidia of Aspergillus niger

We have examined the effects of the enzyme inhibitors 2,4,6-trinitrobenzene sulfonic acid (TNBS) and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) on ethylene and CO₂ production in apple and tomato fruit discs. In the past these inhibitors have been used to inhibit membrane bound enzyme systems in various animal tissues. The amino reactive inhibitor TNBS was shown to decrease ethylene production in tomato discs without affecting rates of respiration; similar results were obtained with apple. The effects of the sulfhydryl reactive inhibitor DTNB were not as clearcut as TNBS. There was little effect of DTNB on ethylene production in tomato discs, however, in apple discs ethylene production was significantly reduced. DTNB also reduced the respiration rate in apple discs, although not to the same extent as ethylene production. The inhibition of DTNB was reversed by a brief treatment with dithioerythritol. The results indicate that ethylene production takes place at the cell surface.     

Imaginal wing discs in larvae of the soldier caste of Pheidole bicarinata vinelandica Forel (Hymenoptera : Formicidae)

In Pheidole bicarinata vinelandica, soldier larvae have prominent mesothoracic wing discs. Imaginal wing discs are suppressed in minor worker larvae. In soldiers, wing discs appear abruptly late in larvae life and are unusually large when compared with wing discs in worker larvae of other ant genera. Once development has been initiated, wing discs of soldier larvae grow at a rate comparable to soldier leg discs. The dynamics of development of soldier wing discs differ fundamentally from those of other holometabolous insects, worker ants and Pheidole bicarinata queens. This unusual developmental pattern may provide a clue to the physiological basis and timing of soldier determination.     

Effect of cutting on solute uptake by plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves.

The uptake of sucrose, 3-O-methylglucose (3-O-MeG), and valine were studied in discs and in purified plasma membrane vesicles (PMV) prepared from sugar beet (Beta vulgaris L.) exporting leaves. The uptake capacities of freshly excised leaf discs were compared with the uptake in discs that had been floated for 12 h on a simple medium (aging) and with discs excised from leaves that had been cut from the plant 12 h before the experiments (cutting). After cutting, sucrose uptake amounted to twice the uptake measured in fresh discs, whereas the uptake of 3-O-MeG and valine remained unaffected. In aged leaf discs, there was a general stimulation of uptake, which represented 400, 300, and 400% of the uptake measured in fresh discs for sucrose, 3-O-MeG, and valine, respectively. Sucrose uptake in fresh discs was sensitive to N-ethylmaleimide (NEM), to p-chloromercuribenzenesulfonic acid (PCMBS), and to mersalyl acid (MA). Although cutting induced the appearance of a sucrose uptake system that is poorly sensitive to NEM but sensitive to PCMBS and MA, aging induced the development of an uptake system that is sensitive to NEM but poorly sensitive to PCMBS and MA. Autoradiographs of discs fed with [14C]sucrose show that cutting resulted in an increase of vein labeling with little effect in the mesophyll, whereas aging induced an increase of labeling located mainly in the mesophyll. The data show that cutting is sufficient to induce dramatic and selective changes in the uptake properties of leaf tissues and that the effects of cutting and aging on the uptake of organic solutes are clearly different. Parallel experiments were run with purified PMV prepared from fresh and cut leaves. The uptake of sugars and amino acids was studied after imposition of an artificial proton motive force (pmf). Comparison of the uptake properties of PMV and of leaf tissues indicate that the recovery of the sucrose uptake system in PMV is better than the recovery of the hexose and of the valine uptake systems. As observed with the leaf discs, cutting induced a 2-fold increase of the initial rate of sucrose uptake in PMV but did not affect the uptake of valine and 3-O-MeG. Cutting induced an increase of both Vmax and Km of the sucrose transport system in PMV. Measurements of the pmf imposed on the vesicles indicated that the increase of sucrose uptake induced by cutting was not due to a better integrity of the vesicles. Hexoses did not compete with sucrose for uptake in PMV from fresh and cut leaves, and maltose was a stronger inhibitor of sucrose uptake in PMV from cut leaves than in PMV from fresh leaves. The sensitivity of sucrose uptake to NEM, PCMBS, and MA in PMV from fresh and cut leaves paralleled that described above for the corresponding leaf discs. These data show that (a) the changes induced by cutting on sucrose uptake by leaf discs are due to membrane phenomena and not to the metabolism of sucrose; (b) the study of sucrose uptake with PMB gives a good account of the physiological situation; and (c) the specific effects induced by cutting on the sucrose uptake system are not lost during the preparation of the PMV.     

Pupal commitment and its hormonal control in wing imaginal discs

The timing of pupal commitment of the forewing imaginal discs of the silkworm, Bombyx mori, was determined by a transplantation assay using fourth instar larvae. The wing discs were not pupally committed at the time of ecdysis to the fifth instar. Pupal commitment began shortly after the ecdysis and was completed in 14 h. When the discs of newly molted larvae (0-h discs) were cultured in medium containing no hormone, they were pupally committed in 26 h. In vitro exposure of 0-h discs to 20-hydroxyecdysone accelerated the progression of pupal commitment. Methoprene, a juvenile hormone analog (JHA), did not suppress the change in commitment in vitro at physiological concentrations. Thus the wing discs at the time of the molt have lost their sensitivity to JH, and 20E is not a prerequisite for completion of pupal commitment. These results suggest that the change in commitment in the forewing discs may begin before the last larval molt.     

Relative contribution of surface and edge to ion absorption by leaf discs

Summary Phosphorus (H₃P³²O₄) absorption was studied in detached bean leaves and leaf discs and later computed per unit surface area and per unit edge length vis-a-vis discs of 4–18 mm diameters. Not only absorption through edge predominantly contributed to total disc absorption it was also directly proportional to disc diameter. Part of the work carried out during senior author's (BKG) tenure as IAEA expert at Bogota.     

Phase determination of the circadian rhythm of conidiation in heterocaryons between two out-of-phase mycelia in Neurospora crassa

Neurospora grows vegetatively as a syncytium in which multiple nuclei exist within a connected cytoplasm. Because of the ability of separate and distinct mycelia to fuse, the possibility exists of generating heterocaryotic cultures in which the nuclei and cytoplasms of two different strains are comingled into the same syncytium. We have used such heterocaryons, in which the component parts differed with respect to their circadian clock phase, to examine whether or not clock-dominant phases exist in the circadian cycle. To this end, the phase subsequent to the formation of heterocaryons by pairs of mycelial discs that are initially at different circadian phases was examined in Neurospora crassa. The resulting phase was an average of the parent phases in many cases, but was sometimes observed to correspond more closely to just one of the original parental phases. In these cases, we did not observe any dominant phases in the circadian cycle; the phase of a particular parent disc was more dominant in the heterocaryon when the proportion of the nuclei from that parent was greater in the heterocaryon. In some instances, which occurred mostly when the difference in phase of the parental discs was large, the resultant phase could not be related in a simple way to the parental phases. An interpretation based on a limit cycle model of the circadian oscillation is possible.     

Etude comparéd de l’action de la benzylaminopurine et de l’acide gibbérellique sur l’absorption du phosphate par des disques de feuille dePelargonium zonale (L.) Aiton. Effects de l’âge et de la température

The absorption of³²P-phosphate (KH₂PO₄-0.1 mM) was studied with discs ofPelargonium zonale leaves, in relation to the age of the leaf, to the ageing process and in relation to the influence of GA3 and BAP. The results show that: i) the speed of absorption reaches a maximum which is characteristic of the adult phase (stage); ii) the “ageing” (washing during 24 h) induces a general increase in the absorption velocity which is dependent on the temperature of the ageing medium (optimum at 26 °C). On the other hand, ageing appears to be a process able to increase the physiological differences which are present before “ageing”. This phenomenon also appears in the case of the influence of phytohormones; iii) if BAP exercises a dose-dependent inhibitory action, in contrast, GA₃ acts by increasing the velocity of the absorption which appears to be a process independent of the hormone concentration. These results underline a phytohormonal specificity. All these results are discussed in relation to hormone-directed transport, cell permeability and leaf senescence.     

Studies in the Physiology of Lichens: With two Figures in the Text: The Effects of Starvation and of Ammonia Absorption upon the Nitrogen Content of Peltigera polydactyla

Discs cut from the thallus of the lichen Peltigera polydactylacontain relatively large amounts of ammonia nitrogen and smallamounts of amide nitrogen. During starvation in the dark theamounts of ammonia and amide nitrogen remain unchanged, butthe amount of amino-nitrogen increases. Absorption of ammoniaresults in increases in the amounts of ammonia and amino-nitrogenin the discs, but not in the amount of amide nitrogen. It istherefore concluded that amides do not have an important quantitativerole in the nitrogen metabolism of the discs. Ammonia absorption is stimulated by addition of glucose to themedium, but the very slow rate of protein synthesis is onlyslightly increased by glucose. The rate of nitrate absorptionis much slower than that of ammonia absorption. The ecological implications of the results are discussed, andexisting knowledge of the nitrogen metabolism of lichens isreviewed.     

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.     

The effect of zearalenone on PSII photochemical activity and growth in wheat and soybean under salt (NaCl) stress

The effects of mycotoxin zearalenone (ZEN) on the photochemical activity of photosystem II (PSII) in wheat and soybean leaf discs incubated in ZEN solutions as well as the after-effects of pre-sowing soaking of seeds in solutions containing ZEN on the photochemical activity of PSII and on the seedlings growth under salt stress (NaCl solutions were investigated). The incubation of wheat leaf discs in ZEN solutions strongly inhibited the energy flux per cross section (CS) for absorption (ABS/CS), trapping (TRo/CS) and electron transport (ETo/CS), while the effects of ZEN action on soybean discs were opposite and the values of those parameters significantly increased with the increase in ZEN concentration. Incubation of seeds in a ZEN solution resulted in an increase in photochemical efficiency of PSII in soybean seedlings, but did not induce any response of PSII in those of wheat at medium illuminations. Only at the stronger illumination for both species did ZEN induce an increase in efficiency of excitation energy capture by open PSII reaction centers, photochemical quenching of chlorophyll a fluorescence and quantum yield of PSII electron transport. Pre-sowing soaking of seeds in a ZEN solution decreased the photoinhibitory injuries of PSII in wheat and soybean due to safe scattering of the excess excitation energy through an increase in energy-dependent quenching (qE) and state transition quenching (qT). ZEN when added to NaCl solutions during the period of germination contributed to reduction in the growth inhibition of wheat seedlings. The incubation of wheat leaf discs in ZEN solutions strongly inhibited CS, ABS/CS, TRo/CS and ETo/CS. Possible effects of ZEN on some physiological processes in plants have been discussed especially in the context with photochemical activity of PSII and a salt stress.     

Characterization of Carrot Cell Wall Protein : II. IMMUNOLOGICAL STUDY OF CELL WALL PROTEIN

Antiserum was raised against a synthetic nona-peptide which was predicted to have considerable homology with the unhydroxylated, unglycosylated precursor of cell wall proteins from several plants. The antiserum is able to recognize the major cell wall protein of incubated carrot (Daucus carota) root discs which is produced when the discs are treated with a proline hydroxylase inhibitor and then labeled with radioactive proline. This technique has potential applications in studying cell wall biosynthesis and its regulatory control mechanisms.     

Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment

In rape leaf discs the response to osmotic stress has been found to be associated with increases in putrescine and 1,3-diaminopropane (an oxidation product of spermidine and/or spermine) and decreases in spermidine titers. In contrast, agmatine and spermine titers showed small changes while cadaverine accumulated massively. Similar results were observed in whole rape seedlings subjected to drought conditions. -DL-difluoromethylarginine (DFMA), a specific irreversible inhibitor of arginine decarboxylase, strongly inhibited polyamine accumulation in unstressed rape leaf discs, which suggested that the arginine decarboxylase pathway is constitutively involved in putrescine biosynthesis. In leaf discs treated under high osmotic stress conditions, both DFMA and DFMO (-DL-difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase) inhibited the accumulation of polyamines. Although the stressed discs treated with DFMA had a lower concentration of putrescine than those treated with DFMO, we propose that under osmotic stress the synthesis of putrescine might involve both enzymes. DFMA, but not DFMO, was also found to inhibit cadaverine formation strongly in stressed explants. The effects on polyamine biosynthesis and catabolism of cyclohexylamine, the spermidine synthase inhibitor, aminoguanidine, the diamine-oxidase inhibitor and -aminobutyric acid, a product of putrescine oxidation via diamine oxidase or spermidine oxidation via polyamine oxidase were found to depend on environmental osmotic challenges. Thus, it appears that high osmotic stress did not block spermidine biosynthesis, but induced a stimulation of spermidine oxidation. We have also demonstrated that in stressed leaf discs, exogenous ethylene, applied in the form of (2-chloroethyl) phosphonic acid or ethephon, behaves as an inhibitor of polyamine synthesis with the exception of agmatine and diaminopropane. In addition, in stressed tissues, when ethylene synthesis was inhibited by aminooxyacetic acid or aminoethoxyvinylglycine, S-adenosylmethionine utilization in polyamine synthesis was not promoted. The relationships between polyamine and ethylene biosynthesis in unstressed and stressed tissues are discussed.     

Role of haemagglutinins in adhesion of Erwinia carotovora to potato tissue

The adhesion of two strains each of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica to potato tuber discs, leaflets and tuber cell cultures was examined and found to occur independently of the presence of either mannose-sensitive (MSHA) or mannose-resistant (MRHA) haemagglutinins. Adhesion was generally greater when bacteria were grown in nutrient broth than on phosphate-buffered agar. The specific MSHA inhibitor α-methyl mannoside reduced the adhesion of two strains to tuber discs and leaflets and the specific MRHA inhibitor, asialofetuin inhibited strains only on leaflets. A reduction in adhesion of a MSHA-producing strain by α-methyl mannoside was observed by scanning electron microscopy which found that adhesion was localized at intercellular junctions.