Effect of cycloheximide, gamma irradiation, and phosphorus deficiency on root pressure exudation in tobacco

Cycloheximide drastically reduced the rate of root pressure exudation in detopped tobacco (Nicotiana tabacum L.), and the effect was more pronounced for nitrate salts in the external solution than for some other salts or when the roots were in water. Diurnal periodicity was greatly decreased, and its phase was changed. Effects began within an hour. Cation uptake was reduced by relatively low levels of cycloheximide. The effects of cycloheximide seemed to be reversible. Mild phosphorus deficiency resulted in decreased exudation rates from detopped tobacco and in no response to nitrate. Periodicity of exudation was not greatly affected by phosphorus deficiency, however. Gamma ray irradiation with a ⁶⁰Co source at levels (up to 40-50 kiloroentgens) which are considered disruptive of moderately large molecules had relatively little effect on the exudation rate. Higher levels of irradiation, which disrupt most protein molecules, decreased exudation and obscured periodicity. The results indicate either that new protein (or peptide) synthesis is needed for the rapid nitrate transport or that the deficiency and inhibitor disrupt cellular membranes. Phosphorus deficiency increased the sensitivity of the plants to inhibition by irradiation of the exudation process.     

Transport of sodium into the xylem exudate of tobacco

When tobacco (Nicotiana tabacum L. var. Virginia Gold) plants were pretreated with Na (²²Na) several days before detopping, from 2.3 to 4.9% of Na previously accumulated in roots appeared in the xylem exudate in 7 days after detopping. Na from the external medium, however, was readily transported to the exudate. Moreover, the amount of the pretreatment Na that was transported to the exudate was not influenced by the presence of Na in the external medium. When Na was present in the external medium after detopping, about 4% (with an NaNO₃ post treatment) to 10% (with an NaCl post treatment) of the Na transported to the xylem in the 7 days following detopping originated in the vacuoles. Nitrate salts of K or Na in the external medium after detopping resulted in transport of large quantities of the respective cation to the exudate, but not in increased transport of the pretreatment Na. A much larger percentage of the K that was accumulated after detopping than of the Na similarly accumulated was transferred to the xylem exudate.     

Effect of calcium on transport of cations to the xylem exudate of tobacco

Summary Roots of detopped tobacco plants (Nicotiana tabacum var. Virginia Gold) were exposed to Na, K, and Ca salts or to water, and cation transfer to xylem vessels was measured. In some cases plants had been exposed to Na in addition to regular nutrient solutions before detopping. Calcium in the external medium greatly depressed the transport of Na from the external medium to the xylem vessels and it often stimulated the transfer of K from the external medium to the xylem vessels. The K/Na ratio in the exudate thus was dependent upon the Ca content of the external medium under these conditions. In contrast, externally applied Ca or Ca deficiency had very little effect on the transfer of preaccumulated K and Na from compartments within roots to the xylem vessels. The K/Na ratio in the exudate under these conditions was not related to Ca levels nor to mild Ca deficiency. The ratios decreased with time after detopping regardless of Ca level. Intact plants accumulated more Na than did root systems of detopped plants in a 6-day period.Riverside University of CaliforniaSoil Science and Agricultural Engineering     

Effect of Shoot Removal and Malate on the Activity of Nitrate Reductase Assayed in Vivo in Barley Roots (Hordeum vulgare cv. Midas)

There is a diurnal variation of nitrate reductase activity (NRA) measured in vivo in barley roots (Hordeum vulgare cv. Midas). In intact plants receiving a 16-hour photoperiod, NRA increases when the light is switched on, reaches a maximum value after 7 to 8 hours, and thereafter declines. Shoot removal (detopping) at the start of the photoperiod prevents the rise in NRA; detopping after 5 hours light leads to a rapid fall in NRA. The inclusion of 10 millimolar malate in the external medium causes a rise in NRA in plants detopped at the beginning of the photoperiod and thus seems to substitute partially for the illuminated shoot. Oxalate, fumarate, and tartrate did not have this effect. Preincubation of the roots of intact plants with 10 millimolar malate for 3 hours, prior to detopping, causes an increase in the flux of amino acids into the xylem sap of detopped roots.     

Nitrogen Fixation in Peanut Nodules during Dark Periods and Detopped Conditions with Special Reference to Lipid Bodies

The peanut plant (Arachis hypogaea L.), unlike other known legumes, can sustain nitrogen fixation when prolonged periods of darkness or detopping curtail the supply of photosynthate to the nodule. This ability to withstand photosynthate stress is attributed to the presence of lipid bodies in infected nodule cells. In both dark-treated and detopped plants, the lipid bodies show a gradual decrease in numbers, suggesting their utilization as a source of energy and carbon for nitrogen fixation. Lipolytic activity can be localized in the lipid bodies, and the existence of β-oxidation pathway and, glyoxylate cycle is shown by the release of ¹⁴CO₂ from ¹⁴C lineoleoyl coenzyme A by the nodule homogenate.     

Drought Stress,Permeability to O2 Diffusion,and the Respiratory Kinetics of Soybean Root Nodules

In legume nodules, treatments such as detopping or nitrate fertilization inhibit nodule metabolism and N2 fixation by decreasing the nodule's permeability to O2 diffusion, thereby decreasing the infected cell O2 concentration (Oi) and increasing the degree to which nodule metabolism is limited by O2 availability. In the present study we used nodule oximetry to assess and compare the role of O2 limitation in soybean (Glycine max L. Merr) nodules inhibited by either drought or detopping. Compared to detopping, drought caused only minor decreases in Oi, and when the external O2 concentration was increased to raise Oi, the infected cell respiration rate in the drought-stressed plants was not stimulated as much as it was in the nodules of the detopped plants. Unlike those in detopped plants, nodules exposed to moderate drought stress displayed an O2-sufficient respiration rate that was significantly lower than that in control nodules. Despite possible side effects of oximetry in altering nodule metabolism, these results provided direct evidence that, compared to detopping, O2 limitation plays a minor role in the inhibition of nodule metabolism during drought stress and changes in nodule permeability are the effect, not the cause, of a drought-induced inhibition of nodule metabolism and the O2-suffiecient rate of respiration.     

Xylem exudation is related to nitrate assimilation pathway in detopped maize seedlings: use of nitrate reductase and glutamine synthetase inhibitors as tools

The xylem exudation of detopped 7-d-old seedlings of Zea maysL. doubled when KCI was present in the root medium comparedto seedlings maintained on water. It was further enhanced whenKCI was replaced by nitrogen compounds such as nitrate, ammoniumand glutamine. The role of the nitrate assimilation pathwayon the enhancement of xylem exudation rate was investigatedusing tungstate, an inhibitor of nitrate reductase (NR) activity,and phosphinothricin or methionine sulphoximine, inhibitorsof glutamine synthetase (GS) activity. The sap levels of NO₃^–,NH₄⁺, glutamine, and asparagine was used to ascertain the invivo inhibition of both enzymes. The tungstate effects werealso checked by measuring leaf in vitro NA activity and NR proteincontent. Xylem exudation rate of detopped seedlings fed withKNO₃ decreased when the nitrate assimilation pathway was blockedeither at the NR or at GS sites. This decrease was preventedwhen urea (acting as NH₄⁺ supply) was given simultaneously withtungstate. KNO₃ does not act directly on exudation, but throughthe involvement of NH₄⁺. The involvement of glutamine was alsoshown since GS inhibition resulted in a cancellation of theenhancing effect of KNO₃ on exudation. As change of exudationrate was not linked to change in sap osmolarity, it is assumedthat the assimilation chain could modify root water conductance.The role of glutamine was discussed. Key words: Exudation, maize, nitrate, conductance, NR, GS     

Ion supply capacity of roots in relation to rejuvenation of primary leaves in vivo

Removal of the shoot above the primary node (detopping) of 3-week-old bean plants ( Phaseolus vulgaris L. cv. Contender) altered the metabolism and development of the remaining leaves. An increase in levels of chlorophyll, protein, stomatal opening, photosynthesis and growth, i.e. rejuvenation of primary leaves, was established within 7 days of detopping. These levels were maintained while the primary leaves of equivalent intact plants senesced.
The flux of xylem solution (mineral ions, cytokinins and water) into leaves is related to the leaf area to be supplied and root supply capacity; it has been suggested that detopping leads to an increased availability of root-supplied solutes and hence rejuvenation of the remaining leaves. This assumes however that root output of solutes is not decreased by the defoliation treatment.
We found that root output of ions (electrical conductivity of passive xylem exudate) in detopped plants was 30% lower than in intact plants after 24 h and 60% lower after 7 days. The output of Ca²⁺, Mg²⁺ and K⁺ were similarly reduced 7 and 14 days after detopping as were fresh and dry weights of roots. Furthermore, neither the calculated xylem flux of ions nor directly measured levels of Ca²⁺, Mg²⁺ and K⁺ were significantly increased in leaves of detopped plants during their rejuvenation. We therefore conclude that root output is tightly coupled to shoot demand and that the apparent rejuvenation of primary leaves caused by detopping bean plants is not a consequence of increased xylem flux of mineral ions into the leaves.     

Effect of nitrogen salts on nitrate reductase activity and protein contents in wheat (Triticum aestivum L.)

The effects of different nitrogen salts on nitrate reductase activity and protein contents were investigated in three Yugoslav cultivars of wheat. The nitrate salts appeared to be a better form of nitrogen than ammonium in respect of the increase of the nitrate reductase activity and root total protein contents, whereas the treatment with ammonium salt resulted in a comparably higher shoot total protein contents. KNO₃ was the best in respect of the level of nitrate reductase activity. Different concentrations of nitrate and ammonium ions in nutrient solution, showed very similar effects on investigated parameters. NS Rana 2 cultivar had the highest values of nitrate reductase activity and protein contents.     

Day-night periodicity of exudation in detopped tobacco

Exudate was collected periodically from the root systems of detopped tobacco plants. Volume, cations, and ⁴²K or ⁸⁶Rb transfer were measured. According to measurements of K by ⁴²K and by the flame photometer, when concentrations of KCl and KNO₃ were lower than 10⁻² m, the K in the exudate came mostly from a pool in the plant rather than from the external solution. With higher external KNO₃ solutions, within a few hours nearly all of the K in the exudate came directly from the external solutions. Studies with ⁸⁶Rb lead to the same conclusion. In contrast the maximum proportion of K in the exudate that came from KCl in the external solution was reached usually in many hours after detopping and amounted to from 50 to 75%. The higher the external concentration the faster it was reached. These data for KCl are indicative of the ⁴²K passing through a K pool in the root cells. K and Rb from high concentrations of KNO₃ and RbNO₃, however, may not pass through such a pool. The addition of 10⁻² m KNO₃ into the external solution during exudation essentially eliminated the effect of periodicity at least for a period of time and under the conditions of the experiments. Hydrochloric acid, mercuric chloride, anaerobiosis, and 2,4-dinitrophenol had the same effect and each resulted in a massive final exudation that usually persisted for 1 to 3 days before stopping. These results all lead to a hypothesis that periodicity is regulated at the tonoplast.     

Influence of CO2 on Reduction of Nitrate in Corn Seedlings

The influence of CO₂ on the assimilation of nitrate in intact corn seedlings was measured with ¹⁵N labelled nitrate, 24 and 48 h after the dark-grown seedlings were transferred to the light, either in normal air or in CO₂-free air. During the first 24 h CO₂ had no influence on nitrate reduction in intact seedlings. Experiments with detopped seedlings showed that during this period the roots were the only site of nitrate reduction. After 48 h seedlings grown in normal air had reduced more nitrate than detopped seedlings, and seedlings grown in CO₂-free air had reduced the same amount of nitrate as detopped seedlings. During the whole 48 h period CO₂ had no influence on the level of nitrate reductase of the leaves. It was concluded that in normal air corn leaves started to reduce nitrate after a lag period of 24 h and that in CO₂-free air they were incapable of nitrate reduction.     

Nitrate Assimilation during Vegetative Regrowth of Alfalfa

Dry matter accumulation, nitrate reductase activity of various organs, nitrate accumulation, nitrogen derived from nitrate, and nitrogen content were studied during 17 days of vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.). Seedlings were grown in the glasshouse and treated with 0, 40, and 80 kilograms N per hectare applied as K¹⁵NO₃ to determine whether reduced nitrogenase activity after shoot harvest limited vegetative regrowth. The role of nodules in reducing NO₃⁻ during this period of low nitrogenase activity was also investigated.     

Detopping causes production of intercellular space occlusions in both the cortex and infected region of soybean nodules

A structural analysis was conducted to determine whether glycoprotein‐containing intercellular space occlusions are involved in medium‐term regulation of O₂ diffusion in soybean (Glycine max) nodules. Alterations in O₂ diffusion were induced by a 3 h detopping treatment, and glycoprotein was immunolocalized with the monoclonal antibodies MAC236 and MAC265. Western blots of unstressed nodules revealed that these antibodies recognize antigens with two different molecular weights in soybean nodules. Tissue printing of halved nodules showed that both antigens were present in fresh nodules from control and 3 h detopped plants. The main localization appeared to be the inner cortex, but some immunolabelling also occurred in the infected region. ELISAs demonstrated a significant increase in total nodule concentration of intercellular glycoprotein following detopping, and cryosections of fresh nodules from this treatment also showed localization of antigens within the intercellular spaces of the infected region. The production of intercellular space occlusions in both the mid‐cortex and infected regions after 3 h detopping was confirmed by light microscopy and silver‐enhanced immunolabelling; cortical changes were quantified by image analysis techniques. Electron microscopy revealed that the occlusions within the infected region were less dense and less heavily labelled than those in the cortex. These results are discussed in relation to O₂ diffusion regulation in soybean nodules     

Studies on Genetic Male-Sterile Soybeans : IV. Effect of Male Sterility and Source of Nitrogen Nutrition on Accumulation, Partitioning, and Transport of Nitrogen

Soybean (Glycine max [L.] Merr.) germplasm, isogenic except for loci controlling male sterility (ms₁) and nodulation (rj₁), was used to investigate the effects of reproductive tissue development and source of nitrogen nutrition on accumulation, transport, and partitioning of nitrogen in a greenhouse experiment. Nodulated plants were supplied nitrogen-free nutrient solution, and nonnodulated plants were supplied nutrient solution containing 20 millimolar KNO₃. Plants were sampled from flowering until maturity (77 to 147 days after transplanting).

Accumulation rates of nitrogen in whole plants during reproductive growth were not significantly different among the four plant types. Nitrogen accumulation in the sterile, nonnodulated plants, however, ceased 2 weeks earlier than in fertile, nonnodulated or fertile and sterile, nodulated plants. This early cessation in nitrogen accumulation resulted in sterile, nonnodulated plants accumulating significantly less whole plant nitrogen by 133 days after transplanting (DAT) than fertile, nonnodulated plants. Thus, changing the site of nitrogen assimilation from nodules (N₂-fixing plants) to roots and leaves (NO₃-fed plants) resulted in similar whole-plant nitrogen accumulation rates in fertile and sterile plants, despite the absence of seed in the latter.

Leaflet and stem plus petiole tissues of both types of sterile plants had significantly higher nitrogen concentrations after 119 DAT than both types of fertile plants. Significantly higher concentrations and exudation rates of nonureide, reduced-nitrogen in xylem sap of sterile than of fertile plants after 105 DAT were observed. These latter results indicated possible cycling of nonureide, reduced-nitrogen from the downward phloem translocation stream to the upward xylem translocation stream in roots of sterile plants. Collectively, these results suggest a lack of sinks for nitrogen utilization in the shoots of sterile plants. Hence, comparison of nitrogen accumulation rates for sterile and fertile plants does not provide a definitive test of the hypothesis that reproductive tissue development limits photosynthate availability for support of N₂ fixation and nitrate assimilation in determinate soybeans.

Nitrogen assimilation during reproductive growth met a larger proportion of the reproductive-tissue nitrogen requirement of nitrate-dependent plants (73%) than of N₂-fixing plants (63%). Hence, vegetative-tissue nitrogen mobilization to reproductive tissue was a more prominent process in N₂-fixing than in nitrate-dependent plants. N₂-fixing plants partitioned nitrogen to reproductive tissue more efficiently than nitrate-dependent plants as the reproductive tissues of the former and latter contained 65 and 55%, respectively, of the whole-plant nitrogen at the time that nitrogen accumulation in reproductive parts had ceased (133 DAT).

     

Nitrogen status of cotton subjected to two short term periods of waterlogging of varying severity using a sloping plot water-table facility

Summary Cotton is reported to be susceptible to waterlogging, and there is evidence that some of the symptoms shown by waterlogged plants are due to impaired uptake of nitrogen. To investigate this for cotton, the nitrogen nutrition of a field-grown crop was monitored when the plants were subjected to two short term periods of waterlogging of varying severity using a sloping plot water-table facility. Growth of severely waterlogged cotton decreased after 4 days in the first and second floodings, and these plants were wilted by the end of the first flooding but not the second. Waterlogging resulted in decreased concentrations of total-N and especially NO ₃ ^– –N in the petiole and lamina of the youngest fully-expanded leaf. Uptake of N by waterlogged plants occurred, but was not as great as for well-aerated plants. The nitrate reductase activity of leaves was much lower in waterlogged plants. Stumps of detopped waterlogged plants did not exude sylem sap at the end of the first flooding, suggesting impaired solute uptake due to damaged roots. However, xylem exudate was obtained from stumps of waterlogged plants at the end of the second flooding, indicating adaptive changes to the root systems of these plants. Although cotton is reported to reduce little NO ₃ ^– –N in its roots, analysis of xylem exudate showed that about half of the N exported by roots was as amino compounds. The concentration of amino compounds in xylem exudate from severely waterlogged plants was higher than in well-aerated plants. It was concluded that the growth reduction in waterlogged cotton was due partly to induced N-deficiency.     

Seedling Nitrate Reductase Activity and Nitrate Partitioning in Maize (Zea mays L.) Strains Divergently Selected For Post-anthesis Leaf-Lamina Nitrate Reductase Activity

Two experiments were conducted to evaluate the effects of phenotypicrecurrent selection for high and low post-anthesis leaf-laminain vivo NRA on nitrate uptake, nitrate partitioning and in vitroNRA of seedling roots and leaves. In Experiment 1, intact plantsof cycle 0, 4, and 6 of the high and low NRA strains were grownon NH₄-N for 11 d, then exposed to 1.0 mol m^–3 KNO₃, andcultures sampled at 6 h and 28 h (induction and post-inductionperiods). Nitrate uptake, tissue nitrate concentration and invitro NRA were determined. The pattern of response to selectionin seedling leaf NRA was similar to that observed for in vivoNRA of field grown plants. Leaf NRA increased between 6 h and28 h. Root NRA was not affected by selection or sampling time.Treatments differed in total fresh weight but not in reductionor uptake of nitrate per unit weight, indicating a lack of correspondencebetween NRA and reduction and supporting the idea that concomitantreduction by NR is not obligatorily linked to nitrate influxin the intact plant. In Experiment 2, dark-grown plants of cycle 0, and 6 of thehigh and low NRA strains were cultured without N, detopped onday 6, transferred the following day to 0-75 mol m^–3 KNO₃and sampled at 6 h and 28 h. In contrast to Experiment 1, selectionpopulations differed in nitrate reduction and root NRA, whichby 28 h reached higher average levels than root NRA of intactplants. Translocation and reduction were inversely related amongstrains within each sampling time. The high level of translocationin detopped plants of the low NRA strain was difficult to reconcilewith its low leaf NRA level of Experiment 1. It is suggestedthat nitrate transport in detopped roots is altered relativeto the intact system in a way which permits greater NRA inductionand nitrate reduction. The results indicate that nitrate partitioningby detopped root systems should be interpreted with caution. Key words: Zea, nitrate reductase activity, nitrate uptake, nitrate reduction, nitrate partitioning, selection     

Day-night Differences in the Accumulation and Translocation of Ions by Tobacco Plants

More K⁴², Rb⁸⁶, and Br⁸² was accumulated by intact tobacco plants and root systems from detopped plants during the night than during the day when studies for both periods were made in dark rooms controlled at 18°. More, however, was translocated to shoots during the day than during the night in intact plants. This latter effect parallels the periodicity of exudation in root systems from detopped plants.     

Glycolytic Flux Is Adjusted to Nitrogenase Activity in Nodules of Detopped and Argon-Treated Alfalfa Plants

To investigate the short-term (30–240 min) interactions among nitrogenase activity, NH₄⁺ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O₂ (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O₂-treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O₂-treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.     

The Role of Oxygen in the Regulation of Nitrogenase Activity in Drought-Stressed Soybean Nodules

The aim of this study was to investigate the mechanism of nitrogenase inhibition in drought-stressed soybean (Glycine max L.) nodules to determine whether this stress was similar to other inhibitory treatments (e.g. detopping) known to cause an O2 limitation of nodule metabolism. Nodulated soybean plants were either detopped or subjected to mild, moderate, or severe drought stress by growth in different media and by withholding water for different periods. All treatments caused a decline in nitrogenase activity, and in the drought-stressed nodules, the decline was correlated with more negative nodule water potentials. Increases in rhizosphere O2 concentration stimulated nitrogenase activity much more in detopped plants than in drought-stressed plants, reflecting a greater degree of O2 limitation with the detopped treatment than with the drought-stressed treatment. These results indicated that drought stress differs from many other inhibitory treatments, such as detopping, in that its primary cause is not a decrease in nodule permeability and a greater O2 limitation of nodule metabolism. Rather, drought stress seems to cause a decrease in the maximum O2-sufficient rate of nodule respiration or nitrogenase activity, and the changes in nodule permeability reported to occur in drought-stressed nodules may be a response to elevated O2 concentrations in the infected cell that may occur as nodule respiration declines.     

RNA Synthesis in Phaseolus Chloroplasts: II. RIBONUCLEIC ACID SYNTHESIS IN CHLOROPLASTS FROM DEVELOPING AND SENESCING LEAVES

The rate of RNA synthesis in chloroplasts from the primary leavesof Phaseolus vulgaris L. cv. Canadian Wonder was measured invitro as plant age increased. The rate per leaf began to fallbefore the leaf was 70% expanded. At full expansion, activityhad fallen by 70%. Chloroplast RNA synthesis per unit chlorophyllwas falling before the leaf was 25% expanded. When all parts of the plant above the mature primary leaveswere removed (detopping) chloroplast RNA synthesis in theseleaves rose within 36 h. The rate increased to a maximum 3–4d after detopping, when it was 5–10 times control values;thereafter it fell again. The chlorophyll content began to increaseabout 4 d after detopping, eventually rising by 100%. Detoppingcaused a 3-fold increase in the Triton X-100-soluble DNA contentof chloroplast preparations, measured after 3.5 d. At that timethe rate of RNA synthesis per unit Triton-soluble DNA was thesame in chloroplasts from the primary leaves of intact and detoppedplants. Detopping also resulted in an increase in the depthof the leaf palisade layer. The effects of detopping on chloroplasts were prevented by darknessand reduced by shading. Increased chloroplast RNA polymerase activity was also inducedin the primary leaves by placing a polythene bag over intactplants, enclosing everything above these leaves. Removal ofthe roots from detopped plants prevented the rise in the rateof chloroplast RNA synthesis.