Effect of Nitrogen Supply on the Growth and Senescence of Leaves of Lolium perenne with Contrasting Rates of Leaf Respiration

Respiration rates remain high during senescence which is anenergy demanding process. Different rates of senescence mayexplain the contrasting respiration rates of mature leaves oftwo populations of perennial ryegrass cv. S23. This hypothesiswas tested by measuring the lengths of elongating and senescingleaves of plants of two populations (GL72, a slow and GL66,a fast respiring population) for 76 d following the transplantationof the seedlings into soil-filled pots in a growth room. Nitrogenwas supplied at two different rates at the beginning of theexperiment and again at day 36. At high levels of nitrogen supply the slow respiring populationhad a faster elongation rate and so a greater leaf length thanthe fast respiring population. There was no difference betweenthem in the rate of leaf senescence or in the duration of leafgrowth. Consequently, the fast respiring population is consideredprofligate in its use of carbon. By contrast, under low nitrogensupply both elongation and senescence rates were higher in thefast respiring population, although there was no differencein final leaf length. Therefore the longevity of leaves of thefast respiring population was reduced. It is argued that theleaves of the fast respiring population may turn-over mineralsmore rapidly than those of the slow, which may be more advantageousin conditions of low nitrogen supply and offset the benefitsof a more conservative use of carbon normally seen under conditionsof high nitrogen supply. Lolium perenne L., perennial ryegrass cv. S23, nitrogen supply, respiration rate, leaf senescence rate, leaf elongation rate, leaf longevity, population     

The effect of handling on photosynthesis, transpiration, respiration, and nitrogen and carbohydrate content of populations of Lolium perenne

Two populations of perennial ryegrass ( Lolium perenne L.) S23, selected for contrasting rates of yield and mature leal dark respiration, were used in this study. Since previous work showed that yield decreased severely in population GL66 due to mechanical perturbation, possible effects on respiration rates were examined. Apart from a transient increase in population GL72, there was no effect on the respiration rate but mechanical perturbation did affect other processes. Handling caused transpiration ratesinthe light to increas for GL72, and in the dark to decrease for GL66. In the logger term, the nitrogen content decreased in handled plants grown at high density. These results emphasize that handling plants should be reduced to a minimum since it might have major effect on several processes. It is concluded that the original selection for the Lolium population was indeed on contrasting rates in yield and respiration. Furthermore it is argued that the existence of low- and high-yielding genotypes can be attributed to the dissimilar responses of different genotypes to mechanical influence.     

Respiration in mature leaves of Lolium perenne as affected by nutrient supply, cutting and competition

The rate of oxygen uptake was measured on sections of mature leaf tissue derived from two populations of Lolium perenne L. cv. S23, selected for contrasting rates of mature leaf respiration. Under conditions of sufficient nutrient supply, GL72 had a lower rate of respiration and a greater yield than GL66. The populations were exposed to important management variables to investigate their effect on the selection criterion, the two variables being nutrient supply and defoliation. Conditions of low nutrient supply were characterized by a similarity in respiration rate and a change in the regulation of respiration. Enzyme levels, rather than adenylates, controlled respiration. Weekly cutting induced activity of the otherwise latent alternative pathway.     

Nitrogen deficiency increases the residence time of respiratory carbon in the respiratory substrate supply system of perennial ryegrass

Plant respiration draws on substrate pools of different functional/biochemical identity. Little is known about the effect of nitrogen deficiency on those pools' sizes, half-lives and relative contribution to respiration, and consequently, of carbon residence time in respiratory metabolism. Here we studied how nitrogen fertilization affects the respiratory carbon supply system of shoots and roots of Lolium perenne , a perennial grass. Plants grown at two nitrogen supply levels in continuous light were labelled with ¹³CO₂/¹²CO₂ for intervals ranging from 1 h to 1 month. The rate and isotopic composition of shoot, root and plant respiration were measured, and the time-courses of tracer incorporation into respired CO₂ were analysed by compartmental modelling. Nitrogen deficiency reduced specific respiration rate by 30%, but increased the size of the respiratory supply system by 30%. In consequence, mean residence time of respiratory carbon increased with nitrogen deficiency (4.6 d at high nitrogen and 9.2 d at low nitrogen supply). To a large extent, this was due to a greater involvement of stores with a long half-life in respiratory carbon metabolism of nitrogen-deficient plants. At both nitrogen supply levels, stores supplying root respiration were primarily located in the shoot, probably in the form of fructans.     

Regulation of Respiration in the Leaves and Roots of Two Lolium perenne Populations with Contrasting Mature Leaf Respiration Rates and Crop Yields

Measurements of O₂ uptake were made on leaves and roots of two populations of Lolium perenne L. cv S23 (GL66 and GL72), previously shown to have contrasting rates of CO₂ evolution and yields of dry matter. O₂ uptake was faster in the mature leaves of GL66 than those of GL72, but no difference was observed in the respiratory rates of meristematic leaf bases or mature roots. The growth rate of GL72 was faster than that of GL66. Cyanide resistance was substantial in mature leaves but the alternative path did not contribute to O₂ uptake in the dark. In both populations, adding malate and glycine stimulated O₂ uptake, but exogenous sucrose only stimulated when uncoupler was also present. The difference between the respiratory rates of the two populations was maintained under all investigated conditions. We conclude that the rate of mature leaf respiration in the dark in L. perenne is limited by adenylate control of glycolysis. The difference between the fast (GL66) and slow (GL72) respiring populations reflected a greater respiratory capacity and higher turnover of ATP in GL66. Alternative path capacity was also high in the roots of both and contributed substantially to O₂ uptake, as indicated by inhibition by salicylhydroxamic acid in the absence of KCN. The alternative path capacity of meristematic leaf bases was considerably less than that in mature leaves.

Transverse and cross-sections were made of mature leaves of both populations to study anatomical features which might explain the differences in ATP turnover, suggested by the biochemical experiments. Leaves of GL72 were thicker but did not show a different anatomy when compared with GL66. The increased thickness was not due to more or larger cells but entirely to a larger intercellular volume.

     

Leaf and root respiration of Lolium perenne populations selected for contrasting leaf respiration rates are affected by intra- and interpopulation interactions

Autotoxicity and allelopathy affect the yield of GL66 and GL72, two populations of perennial ryegrass (Lolium perenne L. cv. S23) that were originally selected for contrasting rates of mature leaf dark respiration, but under conditions where allelopathic effects could not occur and autotoxic effects were minimal. To investigate if the respiration properties are also affected, the populations were subjected to two conditions (monoculture and mixed culture) and two treatments (grown in `renewed', i.e. solution completely replaced every week, and `replenished', i.e. nutrients added to the solution every week, to compensate for the nutrients that had been absorbed from the solution). When plants were grown in mixed culture with a `renewed' nutrient solution, the rate of mature leaf respiration in the absence of inhibitors was higher in GL66 than in GL72. In GL66, this rate was unaffected by condition or treatment, whereas in GL72 it increased in the `replenished' cultures, due to autotoxic and allelopathic effects. In contrast with GL66, for GL72 the effect of an inhibitor of the alternative path (salicylhydroxamic acid, SHAM) differed between conditions and treatments. Effects of an inhibitor of the cytochrome path (KCN) were affected by treatment in both populations. The increase in the rate of root respiration of GL66 due to autotoxic and allelopathic effects was accompanied by a higher rate of the SHAM-resistant component, i.e. maximum activity of the cytochrome path. Roots of GL72 showed a higher control respiration rate and a higher rate of the SHAM-resistant component when long-term accumulation of root exudates was allowed to occur in mixed cultures (allelopathy). We conclude that allelochemicals can modify both leaf and root respiration and that there is no consistent correlation between yield and respiration for GL66 and GL72 in the present study. This shows that selection for low rates of mature leaf respiration is not an appropriate method to select for high-yielding cultivars in perennial ryegrass.     

THE EFFECT OF PHOSPHOLIPASE C, PHOSPHOLIPASE A2 AND NEURAMINIDASE ON THE UPTAKE OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN

Abstract— The uptake of [³H]norepinephrine ([³H]NE) and [³H]serotonin ([³H]5-HT) by rat brain synaptosomes is reduced as a result of pretreatment of the synaptosomes with phospholipase C (EC 3.1.4.3) or phospholipase A₂ (EC 3.1.1.4). This effect is not due to inhibition of the Na⁺-K⁺-ATPase but rather is caused by hydrolysis of neuronal membrane phospholipids, mainly phosphatidylcholine, which seem to be important to the uptake.     

Metabolism of Deoxyuridine in Rabbit Brain

Abstract: The metabolism of [³H]deoxyuridine by rabbit brain was investigated in vitro and in vivo . In vitro , brain slices from various regions of brain and from all age groups accumulated [³H]deoxyuridine from artificial CSF. Within the slices, a portion of the accumulated [³H]deoxyuridine was metabolized to [³H]deoxyuridine phosphate, with subsequent conversion to [³H]thymidine phosphate, and ultimately [³H]DNA. The percentage of the [³H]deoxyuridine phosphorylated and subsequently converted into [³H]DNA was highest at birth and declined to adult levels in 3-month-old rabbits. Thymidine, when added to the incubation medium with the [³H]deoxyuridine, was approximately 10 times as potent as unlabeled deoxyuridine in inhibiting the intracellular phosphorylation and conversion of [³H]deoxyuridine to [³H]thymidine phosphate in brain slices. In vivo , 2.5 h after intraventricular injection of [³H]deoxyuridine, over 90% of the [³H]deoxyuridine was cleared from the central nervous system at all ages. However, in both newborn and 3-month-old rabbits, approximately 40 and 12%, respectively, of the ³H remaining in brain was phosphorylated and converted to [³H]thymidine phosphates; and 11 and 4%, respectively, of the ³H remaining in brain was converted to [³H]DNA. These results show that both immature and mature rabbit brain is able to incorporate deoxyuridine into DNA. Thus, all the enzymes involved in this conversion, including thymidylate synthetase (EC 2.1.1.45), are present and active in brain throughout life.     

[3H]Nemonapride and [3H]Spiperone Label Equivalent Numbers of D2 and D3 Dopamine Receptors in a Range of Tissues and Under Different Conditions

Abstract: [³H]Nemonapride and [³H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [³H]nemonapride and [³H]spiperone give markedly different B _max values for preparations of D₂ dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D_2(short), D_2(long), and D₃] in different buffers. B _max values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [³H]spiperone and [³H]nemonapride do not differentiate between different forms or populations of D₂-like receptors.     

Regionally Distinct N-Methyl-D-Aspartate Receptors Distinguished by Quantitative Autoradiography of [3H]MK-801 Binding in Rat Brain

Abstract: Quantitative autoradiography of [³H]MK-801 binding was used to characterize regional differences in N -methyl- d -aspartate (NMDA) receptor pharmacology in rat CNS. Regionally distinct populations of NMDA receptors were distinguished on the basis of regulation of [³H]MK-801 binding by the NMDA antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). CPP inhibited [³H]MK-801 binding in outer cortex (OC) and medial cortex (MC) with apparent K _i values of 0.32-0.48 μ M , whereas in the medial striatum (MS), lateral striatum (LS), CA1, and dentate gyrus (DG) of hippocampus, apparent K _i values were 1.1-1.6 μ M . In medial thalamus (MT) and lateral thalamus (LT) the apparent K _i values were 0.78 μ M . In the presence of added glutamate (3 μ M ), the relative differences in apparent K _i values between regions maintained a similar relationship with the exception of the OC. Inhibition of [³H]MK-801 binding by the glycine site antagonist 7-chlorokynurenic acid (7-ClKyn) distinguished at least two populations of NMDA receptors that differed from populations defined by CPP displacement. 7-ClKyn inhibited [³H]MK-801 binding in OC, MC, MS, and LS with apparent K _i values of 6.3-8.6 μ M , whereas in CA1, DG, LT, and MT, K _i values were 11.4-13.6 μ M . In the presence of added glycine (1 μ M ), the relative differences in apparent K _i values were maintained. Under conditions of differential receptor activation, regional differences in NMDA receptor pharmacology can be detected using [³H]MK-801 binding.     

The effect of handling on the yield of two populations of Lolium perenne selected for differences in mature leaf respiration rate

Two populations of Lolium perenne L. S23 (perennial ryegrass), selected for differences in mature leaf dark respiration, were used in a non-destructive indexing system for individual plants, to determine growth parameters. Population GL66, selected for high respiratory rates and low yield, responded strongly to the indexing treatment, when grown at low plant density. Dry weights of all plant parts decreased strongly, as did dry matter percentages of the leaf blades. At high density this population demonstrated the same trend, but additionally allocation to the shoot increased. In contrast, GL72, selected for low respiratory rates and a high yield, responded only at a high plant density. It is argued that there might be a relation between the dissimilar response of the two populations to mechanical influences and the presence of the genotypes of the low-yielding population in the parent variety. The results also emphasize that non-destructive growth analyses can only be used when their effects on the plants are known.     

Glutamine and Glucose as Precursors of Transmitter Amino Acids: Ex Vivo Studies

Abstract: Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo . Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30-150 min by [U-¹⁴C]glucose, [U-¹⁴C]glutamine, or [³H]glutamine administered alone or together in doublelabel experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 μ M ); tetrodotoxin (1 μ M ) was present in the control medium. Single-label experiments showed that [U-¹⁴C]- glutamine was more effective than [U-¹⁴C]glucose for labelling releasable glutamate and GABA. Double-label experiments showed that [³H]glutamine and [U-¹⁴C]- glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment empbasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.     

Regulation of DOPA Decarboxylase Activity in Brain of Living Rat

Abstract: To test the hypothesis that l -DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [³H]DOPA. The unidirectional blood-brain clearance of [³H]DOPA ( K ^D₁) was 0.049 ml g⁻¹ min⁻¹. The relative DDC activity ( k ^D₃) was 0.26 min⁻¹ in striatum, 0.04 min⁻¹ in hypothalamus, and 0.02 min⁻¹ in hippocampus. In striatum, 3,4-[³H]dihydroxyphenylacetic acid ([³H]DOPAC) was formed from [³H]DA with a rate constant of 0.013 min⁻¹, [³H]homovanillic acid ([³H]HVA) was formed from [³H]DOPAC at a rate constant of 0.020 min⁻¹, and [³H]HVA was eliminated from brain at a rate constant of 0.037 min⁻¹. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, k ^D₃ was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.     

Localization of ornithine decarboxylase and changes in polyamine content in root meristems of Zea mays

Polyamine content and the activity of arginine decarboxylase (EC 4.1.1.19) and ornithine decarboxylase (EC 4.1.1.17) were studied with respect to meristematic activity in primary roots and in developing lateral roots of Zea mays L. (cv. Neve Ya'ar 170) seedlings. Comparative localization of active ornithine decarboxylase and of meristematic activity were determined by labelling roots either with α-[5-¹⁴C]-difluoromethyl ornithine or with [³H]-thymidine, respectively.
Lateral roots were formed during the 72 h post-decapitation period, accompanied by an initial decline in putrescine content and by a significant increase in spennidine con-tent at 48–72 h. High levels of spermidine and lower levels of putrescine were found in the primary root apex as well. A marked increase in ornithine and arginine decarboxylase activity, as measured by ¹⁴CO₂ release, was found during the 72 h post-decapitation period of lateral root development. This increase in ornithine decarboxylase activity was confirmed also by a parallel rise in the incorporation of α-[5-¹⁴C]-difluoromethyl ornithine into trichloroacetic acid-insoluble fractions. Microautoradiographs of longitudinal and cross sections of roots, labelled with α-[5-¹⁴C]-difluoromethyl ornithine, showed that ornithine decarboxylase is localized mainly in the meristematic zones, as evidenced by [³H]-thymidine incorporation. A close correlation between meristematic activity and polyamines was demonstrated in situ , suggesting that polyamine content and biosynthesis may have a role in meristematic activity in corn roots.     

Comparative Analysis of Different Approaches to Investigate Cell Kinetics

Abstract. The potential of different methods to investigate proliferative activity of cell populations was analysed for non-Hodgkin's lymphomas. Cells in S phase and all cycling cells were determined on cell suspensions obtained from fresh lymph node material by [³H]-thymidine autoradiography ([³H]TdR LI), a monoclonal antibody to bromodeoxyuridine (BrdU LI), and the monoclonal antibody Ki67. A good correlation was observed between the values of [³H]TdR LI and BrdU LI ( r _s= 0.90; P < 0.01), [³H]TdR LI and S phase ( r _s= 0.62; P < 0.01) and [³H]TdR LI and Ki67 ( r _s= 0.64; P < 0.01) in individual lymphomas. Using the median values obtained from the different approaches as cut-off points to define slowly and rapidly proliferating tumours, the best agreement was observed between [³H]TdR LI and BrdU LI (91%) and poorer agreements, even though statistically significant, were observed between [³H]TdR LI and S phase (73%) or Ki67 (76%). In conclusion, the kinetic information derived from different approaches was more or less concordant and newly proposed approaches should be directly and carefully verified for their prognostic relevance before using them as alternatives to conventional methods.     

FAST AXOPLASMIC TRANSPORT OF A CALCIUM-BINDING PROTEIN IN MAMMALIAN NERVE

Abstract— Calcium is transported at a fast rate of 410 mm/day in cat sciatic nerve on injection of ⁴⁵Ca²⁺ into the L7 dorsal root ganglia. Nerve segments corresponding to the crest and the plateau regions of transported activity were analyzed by column chromatography on Sephadex G-100 and Biogel A 5m columns and the fast transported ⁴⁵Ca²⁺ found to be bound to a protein of 15,000 dalton. Using [³H]leucine as a precursor, a labeled calcium binding protein (CaBP) was found located at the same position in elution volumes from the columns as was the protein-bound ⁴⁵Ca^{2 +}. The level of [³H]-labeled CaBP in the crest and plateau regions were compared using column chromatography and polyacrylamide gel electrophoresis techniques and approx 3×4 times more [³H]-labeled activity was found in the crest as compared to the plateau. These findings indicate that Ca²⁺ is fast transported in association with the CaBP. The relation of CaBP to the transport filament model of axoplasmic transport and its possible role in nerve are discussed.     

Glutamine--a major substrate for nerve endings

Abstract— Mammalian cortical synaptosomes incubated in the presence of glucose (2.5 MM) plus glutamine (0.5 mM) showed a 30% increase in transmitter amino acid content over controls with glucose alone and a doubling of glutamate release induced by Veratrine or high K⁺. Double-label experiments, i.e. [U-¹⁴C]glucose with [³H]glutamine, and single-label experiments, i.e. [U-¹⁴C]glucose or [U-¹⁴C]-glutamine showed that stimulus-released glutamate was derived principally (80%) from glutamine. Released glutamine-derived glutamate was of higher (x 2) specific radioactivity than its tissue equivalent. Glutamine alone (0.5–0.75 mM) was much less effective than equivalent amounts of glucose alone, in stimulating respiration and maintaining tissue K⁺ levels.     

THE FORMATION OF GLUTAMATE, ASPARTATE AND GABA IN THE RAT RETINA; GLUCOSE AND GLUTAMINE AS PRECURSORS

Abstract— The distribution of the neuroactive amino acids taurine, GABA, glycine, glutamate and aspartate, together with glutamine, have been studied in the rat retina. Peak levels of taurine were found in photoreceptor cells and of GABA and glycine in a retinal fraction enriched in amacrine cells and, synaptic terminals. In vitro , GABA formation from [³H]glutamine and [¹⁴C]glucose was also most prominent in this fraction; at 500 μ m [³H]glutamine was the better precursor.
Observations on metabolism in the photoreceptor cell layer of the tissue suggest an active turnover of glutamate, aspartate and GABA, and show that glutamine may serve as an alternative substrate to glucose here, perhaps via the GABA bypath.     

The Peripheral-Type Benzodiazepine Receptor Ligands [3H]Ro 5-4864 and [3H]PK 11195 Bind to the Retina of Rabbit, but Not of Turtle

Abstract: The binding of [³H]flunitrazepam, [³H]RO 5-4864, and [³H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [³H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [³H]RO 5-4864 and [³H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (K_D values of 24 ± 2.3 and 2.2 ± 0.8 nM, and B_max values of 440 ± 35 and 1,482 ± 110 fmol/mg of protein, respectively). The binding was temperature - and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [³H]RO 5-4864 and [³H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.     

[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.