The influence of radioactive phosphate level on the absorption of phosphate by plants and on the determination of labile soil phosphate

Summary Previous work has suggested that the presence of P³² in fertilizers inhibits the uptake of the applied phosphate from the soil by plants, and also that if the applied phosphate is not incorporated uniformly in the soil there will be preferential uptake from regions of low specific activity. This made it desirable to determine the effect of P³²-level on phosphate uptake and the determination ofL-values in pot experiments in which the labelled phosphate source is added as discrete particles of the phosphate form of an anion-exchange resin.Increasing the level of P³² from 0.05 to 1.25 mo per gram of phosphorus in the added phosphate did not have a significant effect on the fresh weight, dry weight or total phosphorus uptake of the ryegrass crop. The measuredL-value showed a significant increase, about 15 per cent for a five-fold increase in P³² level, on each of the four soil types used, as would be expected if P³² depressed the uptake of labelled fertilizer phosphate.Although a significant effect of P³² was observed this does not invalidate a comparison of soils with respect toL-value.     

广西典型红壤旱地施用钙镁磷肥对玉米产量及其镉累积的影响

在广西典型类型红壤旱地布置玉米磷肥施用量的田间试验,研究不同钙镁磷肥施用量(磷肥Cd含量为0.0651 mg/kg)对玉米产量及地上部Cd累积的影响。结果表明,与不施磷肥处理(CK)相比,施磷肥可分别显著提高春、秋玉米籽粒产量8.2%—13.1%和13.7%—20.0%。高磷(600 kg P2O5/hm2)处理的春玉米秸秆产量比CK显著提高11.4%;施磷处理春、秋玉米秸秆Cd含量分别下降2.7%—45.8%和11.0%—43.6%;而籽粒Cd含量分别下降13.0%—40.6%和9.9%—31.5%,且秸秆和籽粒的Cd含量及累积量均随施磷量的增加而逐渐降低,其中以高磷处理最为显著。玉米秸秆及籽粒Cd累积量在高磷处理下(600 kg P2O5/hm2)分别比低磷处理(75—300 kg P2O5/hm2)降低13.6%—41.5%和8.8%—29.3%。相关分析表明,玉米Cd含量与土壤pH呈显著负相关,与土壤有效Cd含量呈显著正相关。施磷提高土壤pH,而降低土壤有效Cd含量。高量磷肥施用降低土壤Cd的有效性进而降低玉米对Cd的吸收累积。     

Soil solution dynamics and plant uptake of cadmium and zinc by durum wheat following phosphate fertilization

A growth chamber study was conducted to evaluate the effect of application of phosphate fertilizer on soil solution dynamics of cadmium (Cd) and Cd accumulation in durum wheat (Triticum turgidum L. var. durum). Treatments consisted of three phosphate fertilizer sources containing 3.4, 75.2, and 232 mg Cd kg^?1 applied at three rates (20, 40 and 80 mg P kg^?1) plus a no fertilization control. An unplanted treatment at 40 mg P kg^?1 was included to separate the effects on soil solution Cd dynamics of the crop from that of the fertilizer. Soil solution samples were obtained using soil moisture samplers every 10 days after germination. The experimental results indicated that plant biomass significantly increased with P application rates and decreased with increased Cd concentration in the phosphate fertilizers. Total cadmium concentration in soil solution was not consistently affected by phosphate fertilization rate and fertilizer sources, and therefore Cd concentration in the fertilizer. Application of phosphate fertilizer, however, increased the concentration and accumulation of Cd and shoot Cd/Zn ratio, and decreased shoot Zn concentration in durum wheat. Phosphate sources had a marginally significant effect (P?=?0.05) on shoot Cd concentration and did not affect Cd accumulation in durum wheat. Concentration of Cd in soil solution was unrelated to Cd concentration in durum wheat. These results suggest that the immediate increase in Cd concentration and Cd accumulation in durum wheat with phosphate application is due more to competition between Zn and Cd for absorption into plants, enhanced root to shoot translocation and enhanced root development, than to a direct addition effect from Cd contained in phosphate fertilizer. In the short term, application of phosphate fertilizers can increase Cd concentration in the crops, regardless of the Cd concentration of the fertilizer. An optimal P fertilization, possibly in combination with Zn application, may offer an important strategy for decreasing Cd concentration and accumulation in crops.     

On the effects of phosphorus on zinc uptake by barley

Summary The response of barley to phosphate application and the effect of applied phosphorus on the uptake of soil zinc by the crop were tested in pot, Neubauer and incubaticn experiments on four soils differing in native phosphate status.There was a response of the yield to phosphorus application in alluvial soils from Clementina (Ecuador) and Bangla Desh. Barley grown on red soil from Bangla Desh and glacial clay from Uppsala did not show any response to P application.Applied P³² was fixed to a great extent in all the soils studied. But in phosphate-deficient soils, a much higher degree of sorption of P³² was recorded than in phosphate-rich soils. Compared to phosphate-rich soils, the utilization of fertilizer phosphorus is higher than of native soil P in the case of phosphate-deficient soils. It was observed that uptake of Zn⁶⁵ by the crop is counteracted by phosphorus application at the three stages of crop growth studied.This work was financially supported by the International Atomic Energy Agency, Vienna, Austria, and by the National Council for Forestry and Agricultural Research, Sweden.     

P32 distribution in phosphorus fractions of phosphorus-sensitive and -tolerant soybeans

Summary P³² incorporation into various phosphorylated compounds of roots, stems, and leaves was studied with P-sensitive (Clark) and P-tolerant (L9) varieties of soybeans. With increasing P concentration in the pretreatment solution, more P³² was incorporated into the inorganic phosphate fraction, mainly in which form P seemed to be transported from roots to leaves. P³² percentage of inorganic phosphate also showed a continuous decrease with absorption time. The rate of phosphorylation, as manifested by the ratio of P³² percentage or specific activities of the organic acid-soluble P to that of inorganic-P, was higher with the P-tolerant variety than with the P-sensitive one. The rate of RNA synthesis also seemed to be higher with the tolerant variety. Paper No. 7082, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Permeabilization of plant cells: 31P NMR studies on the permeability of the tonoplast

A suspension culture of Catharanthus roseus has been used to study the permeability of cell membranes after treatment with various concentrations of a permeabilizing agent (DMSO). The uptake and release (after permeabilization) of inorganic phosphate (P_i) by cells have been investigated by ³²P radiotracer and non-invasive phosphorus-31 NMR experiments. These studies have demonstrated that measurements of the P_i-efflux from plant cells provide a reliable measure of the permeability of the tonoplast.Abbreviations DMSO dimethylsulfoxide - NMR nuclear magnetic resonance - P_i inorgainic phosphate     

A study of the mechanism of soil-phosphate uptake in relation to plant species

Summary The concept of plant available and unavailable soil phosphate has been examined by growing a range of plant species in soils well mixed with carrier free P³².It was found that although activities of 5–100 µC per kg soil caused changes in dry matter and P uptake, they had no influence on the specific activity per unit dose.Only small and agriculturally insignificant differences have been found in the proportion of soil P to added P³² taken up by the different species in the acid and neutral soils employed. It is considered that such differences as there are may be due to exchange of phosphate between seed and soil. Marked differences, however, occurred in the total phosphate absorbed by the crops.These results support the view that a fraction of the soil phosphate exists in a labile pool which will exchange with added P³² and will maintain a definite equilibrium potential in the soil solution. Plants do not appear to have a means of increasing significantly the size of the labile pool under the experimental conditions described. The disparity, however, between the total phosphate uptake especially in very deficient soils does suggest that certain species are more efficient than others in the absorption of soil phosphate at low potential.     

Quantitative short-term uptake of inorganic phosphate by the Chara hispida rhizoid

Abstract The rhizoid of Chara hispida L. made a small contribution to the uptake of inorganic phosphate under laboratory conditions. At 1 mmol m^?3 phosphate the rhizoid contributed about 4% to the uptake of the whole plant over 4 h. Under these conditions about half of the phosphate taken up by the rhizoid was translocated into the shoot. The rates of uptake and translocation increased with increasing external phosphate concentrations. When the shoot was in darkness, ³²P-translocation from the rhizoid was less than half of that found when the shoot was illuminated. When the rhizoid medium was anaerobic the translocation rate was lower than the rate in aerobic conditions and illumination of the shoot had no effect on the uptake or translocation of phosphate. Translocated ³²P accumulated in the apical growing regions of the plant. This was first noted in the secondary apices nearest to the rhizoids.     

A single plant technique for field studies of distribution of32P-labelled phosphate between plant and soil pools

Summary ³²P-labelled monocalcium phosphate solution was supplied to the root system of individual wheat plants within a field crop two weeks after emergence. Three levels of carrier P, equivalent to 2.5, 5, and 10 kgP ha^–1 were used. The distribution of³²P between shoots and the soil inorganic and organic P fractions was measured after a further six weeks growth.There was no evidence for the incorporation of³²P-orthophosphate into soil organic P fractions in the wheat rhizosphere in the period between germination and mid-tillering.The suitability of the single plant technique to measure plant available P in field soils was assessed by calculating A values from plants grown in soil witth different fertilizer P histories. There was a significant linear relationship between A values and the amount of soil P extractable with 0.5M NaHCO₃ from the different fertilizer treatments. Although the technique is unlikely to given an absolute value for plant available nutrient, it can provide quantitative data for comparative trials with different forms of fertilizer, methods of fertilizer application or amounts of available soil water. The values obtained should be termed comparative A values or A^c values.     

A chemical and biological approach towards the definition of calcareous soils

Summary Several agricultural problems are associated with the presence of certain levels of CaCO₃ in soils. The level of CaCO₃ at which the phosphate fixation becomes an apparent agricultural problem, is thought to be an appropriate margine at which the soil can be considered calcareous. Thus, a set of soil mixtures, ranging in CaCO₃ content from 1 to 96% was prepared and used in a column study to determine the level at which the CaCO₃ fraction becomes a dominant factor controlling. P³² movement and distribution.Increasing the percentage of oolitic sand, in the soil mixture, from 1 to 10% caused a sharp drop in P³² movement with soil solution and any increase in CaCO₃ content above 10% did not show any further drop in P³² movement. The amount of P³² removed with the soil solution was generally low compared to that retained in soil columns. Studying the distribution of P³² in soil columns, after five displacements, has indicated that the migration of P³² from the top soil increased by increasing CaCO₃ from 1 and 2 to 6%. The amount of P³² removed was however retained in lower sections. A very sharp decrease in P³² migration from the top soil was observed when CaCO₃ content was raised from 8 to 10%.A similar picture was shown when the CaCO₃ material used was in clay size fraction. However the sharp increase in phosphate retention in top soil sections took place at CaCO₃ content of 8% rather than at 10%. A limit of 8 to 10% CaCO₃ was proposed as an appropriate margine for defining calcareous soils.     

Effects of vesicular-arbuscular mycorrhiza on the size of the labile pool of soil phosphate

Summary Inoculation of lettuce, onion and clover with VA mycorrhizal fungus (Glomus mosseae) increased plant yields and phosphate uptake in three soils that had been depleted in phosphate. From two soils in which the labile pool of phosphate had been labelled with³²P, the specific activity of plant phosphate was the same whether the plants were mycorrhizal or non-mycorrhizal. In a third soil (Sonning) the specific activity was lower in lettuce and clover when the plants were mycorrhizal. When the experiment was repeated with the same soil under conditions that gave lower growth rates, the specific activity was the same in mycorrhizal and non-mycorrhizal plants. The lower specific activity in lettuce and clover in the first experiment is atributed to greater release of slowly exchanging phosphate (which is not in equilibrium with the added³²P), caused by the high uptake of phosphate by the mycorrhizal plants. When they occur, lower specific activities in mycorrhizal plants may therefore not necessarily indicate a solubilizing effect of the mycorrhiza on soil phosphate.     

In vitro studies of p32 uptake in mouse liver mitochondria

Isolated mouse liver mitochondria were incubated in two types of P³²-labelled sucrose-phosphate buffers. The first contained no added ATP or oxidizable substrate. The second contained added ATP. Samples were taken at specified times, up to 60 minutes, and analyses were made of the mitochondrial TCA-soluble inorganic P³² and the total mitochondrial residue P³¹ and P³². The results of the analyses showed that when the phosphorus inhibition index (the ratio of the amount of incubation inorganic phosphorus to the square of the amount of tyrosine in the mitochondria) was high, inorganic P³² uptake was low and vice versa. In accordance with established data, increased P³² uptake was obtained when ATP was added. ATP was found to stabilize the turnover of TCA-insoluble residue phosphorus as well as to maintain the TCA-soluble orthophosphate pool. These results support findings regarding the inhibitory and controlling effects of incubation medium phosphate in the regulation of inorganic phosphorus uptake.     

Metabolism of 3- and 4-phosphorylated phosphatidylinositols in stomatal guard cells of Commelina communis L.

Within the plant kingdom the stomatal guard cell is presented as a model system of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-mediated signal transduction. Despite this it is only recently that the phosphoinositide components of animal signal transduction pathways have been identified in stomatal guard cells. Interestingly, stomatal guard cells contain both 3- and 4-phosphorylated phosphatidylinositols though their relative contributions to signalling remain undefined. An appraisal of the routes of synthesis and rates of turnover of these phosphatidylinositols would appear timely as the in vivo biosynthesis of these components is a much neglected facet of the phosphoinositide-mediated signalling paradigm as purported to apply to plants. A non-equilibrium [³²P]P_i labelling strategy and enzymic and chemical dissection of labelled phosphatidylinositols have been used to address not only the route of synthesis but also the rates of turnover of phosphatidylinositols in stomatal guard cells of Commelina communis L. The specific activity of the ATP pool of isolated guard cells was found to increase over a 4 h period when labelled from [³²P]P_i. In separate experiments, isolated guard cells were labelled over a 40–240 min period, their lipids extracted, deacylated and resolved by HPLC. Glycerophosphoinositol phosphate (GroPInsP) and glycerophosphoinositol bisphosphate (GroPInsP₂) peaks were desalted and enzymically cleaved with alkaline phosphatase and human erythrocyte ghosts, respectively. The monoester phosphate in phosphatidylinositol 4-monophosphate (PtdIns4P) accounted for 90–97% of the [³²P]P_i label while the 4- and 5-monoester phosphates of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] accounted for typically 39% and 61% respectively. Therefore, the evidence is consistent with synthesis of PtdIns(4,5)P₂ by successive 4- and 5-phosphorylation of phosphatidylinositol (PtdIns). This study therefore represents the first report of the pathway of the synthesis of 4- and 5-phosphorylated phosphatidylinositols in a single defined hormone-responsive plant cell type. The monoester phosphate in phosphatidylinositol 3-monophosphate (PtdIns3P) accounted for 83–95% of the ³²P label. It was not possible, however, to determine the route of synthesis of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P₂] owing to the rapid attainment of equilibrium between the 3- and 4-monoester phosphates of PtdIns(3,4)P₂, each containing approximately 50% of the label at just 40 min of labelling. Turnover of PtdIns3P was quicker than that of PtdIns4P. Similarly, turnover of PtdIns(3,4)P₂ was quicker than that of PtdIns(4,5)P₂, and in mass terms PtdIns(3,4)P₂ appeared to predominate over PtdIns(4,5)P₂. By analogy with animal systems, in which signalling molecules such as PtdIns(4,5)P₂ show considerable basal turnover, the evidence presented is consistent with signalling roles for PtdIns3P and PtdIns(3,4)P₂ in addition to those previously indicated for PtdIns(4,5)P₂ in stomatal guard cells.     

The supply of nutrient ions by diffusion to plant roots in soil

Summary Measurements were made of the diffusion of P³²-labelled phosphate to single roots of onion, leek and rye-grass growing in an Upper Greensand sandy loam (UGS) and a Coral Rag Clay (CRC) to which different amounts of phosphate had been added. Concentration-dependent diffusion coefficients for phosphate ions in the soils were calculated from phosphate desorption isotherms in calcium chloride. The experimental uptake by roots of known dimensions was compared with supply expected by diffusion to a cylindrical model root of the same dimensions. Allowance was made for absorption by the root hairs on rye-grass roots. Phosphate absorption by a cm length of intact root was found to continue for at least 16 days for onion, 10 days for leek and 5 days for rye-grass. Over a wide range of conditions (phosphate concentrations, soils, plant species), experimental uptake was close to the maximum calculated to be possible for the diffusion model except on one soil at a high level of phosphate. Although the concentration of phosphate in the soil solution at the root boundary appeared to be reduced to a small fraction of the initial concentration, because of the extreme non-linear form of the desorption isotherm less than 1/2 of the P³² exchangeable pool of P was considered to contribute to diffusion. Phosphate uptake by rye grass could only be accounted for if the root hairs were active. Although only a small fraction of the uptake is derived from inside the root hair cylinder, this increases the efficiency of the central root 2.3 fold by providing a zone close to the central root through which phosphate moves very readily.     

Uptake of phosphate from soils and fertilizers as affected by soil P availability and solubility of phosphorus fertilizers

Fertilizers labelled with ³²P were used to measure amounts of phosphorus, P_s and P_F, taken up by Lolium perenne from available soil P and from P fertilizer respectively, when applied at a rate of 66 mg P·(kg soil^–1) in greenhouse experiments. The quantity P_s of phosphorus taken up from soil in the presence of P fertilizer was compared to the quantity P_o taken up from soil without P fertilizer. The quantity (P_s–P_o) is positive for low P_o values, i.e. in soils poor in available phosphorus, but is negative for high P_o values indicating that an input of P fertilizer can induce a decrease in the utilization of available soil phosphorus. Moreover, for a given soil, the quantity (P_s–P_o) depends on the chemical form of the fertilizer. The standard method of evaluation of P fertilizer efficiency is based on the assumption that P_s=P_o, but P_s can differ from P_o. This result can explain the contradictory data published from field experiments about the efficiency of the various P fertilizers.     

Native soil and fresh fertilizer phosphorus uptake as affected by rate of application and P fertilizers

³²P labelled fertilizers were used to measure native soil and fresh fertilizer phosphorus uptake byLolium perenne L. in greenhouse experiments. The P source evaluation was carried out for multiple rates of application for a standard P fertilizer (DAP) on low and medium soil P levels and for North Carolina rock phosphate (RP) at the medium soil P level only. At the low soil P level, the native P uptake increase was independent of P-DAP applied, and represented 19% of the nil P uptake. At the medium soil P level, the variability of the native soil as a nutrient P source depended on the phosphate fertilizer applied, and the rate of application. Consequently the amount of total P uptake could conceal differences in P fertilizer evaluations as the nutrient P source. Fresh P uptake increased linearly with the rates of P applied as standard or tested P fertilizer. The comparison of various P sources by means of fresh P uptake ratio (i.e. fresh P uptake from tested phosphate divided by fresh P uptake from standard phosphate) was independent of the rate of application. It was therefore suggested that various phosphorus sources be evaluated by measuring the fresh P uptake for a single rate of application.     

Effect of 4,6-Dinitro-o-sec-butylphenol on Phosphorus Accumulation and Incorporation in Tomato Leaf Disks

The accumulation and incorporation of externally applied P³² into ATP and the effect of 4,6-dinitro-o-sec-butylphenol (DNBP) on these processes was studied, using tomato (Lycopersicon esculentum Mill.) leaf disks.

P³² was, in most part, actively accumulated into leaf disks with time and was incorporated into ATP and other organic phosphates. DNBP inhibited both P³² accumulation and ATP generation. The amount of inhibition increased with time of incubation.

It is concluded that P³² accumulation is related to ATP generation. Even though DNBP greatly inhibits phosphorus accumulation, there is little or no effect on its retention.

DNBP has the ability to uncouple oxidative phosphorylation. Therefore, it is assumed that its inhibitory effect on phosphate accumulation and generation of high-energy phosphorus esters is related to its inhibition of oxidative phosphorylation.

A method is described which appears to be satisfactory to determine the relative amounts of ATP and ADP in leaf disks labeled with P³².

     

Rapid and efficient method for analyzing phosphorylation of the S6 ribosomal protein in 32Pi-labeled,tissue culture cells

We describe a method for studying the phosphorylation of the S6 ribosomal protein in intact cells. The procedure has the advantage of using few cells, little ³²P_i, and by using an air-driven centrifuge, many samples can be processed in a short time. Metabolically labeling the ribosomes with [³H]uridine before the experiment provides a measure of ribosome yield. The amount of ³²P_i incorporated into proteins other than S6, which cosediment with the ribosomes, increases by the same amount as the specific activity of [³²P]ATP increases, when the cells are stimulated by prostaglandin F_2α, insulin, epidermal, or fibroblast growth factor, or serum; whereas the ³²P_i incorporated into S6 increases by a factor greater than the increase in the specific activity of [³²P]ATP. We show that the phosphate on S6 turns over at least as rapidly as does the phosphate on ATP. This last observation allows us to use a procedure, which we have outlined for determining the absolute amount of phosphate added to S6 due to a stimulus.     

Properties of Bound Inorganic Phosphate on Bovine Mitochondrial F1F0-ATP Synthase

Beef-heart mitochondrial F₁F₀-ATP synthase contained six molecules of bound inorganic phosphate (P_i). This phosphate exchanged completely with exogenous ³²P_i when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by ³²P_i when the enzyme was not pretreated with DMSO. These two molecules of ³²P_i were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound ³²P_i remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of ³²P_i were displaced by ATP. The ATP-resistant ³²P_i was removed from the enzyme by pyrophosphate. It is proposed that these molecules of ³²P_i are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of ³²P_i to DMSO, followed by removal of the DMSO, resulted in the loss of the bound ³²P_i and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a P_i ATP exchange reaction with bound P_i The presence of two catalytic sites containing bound P_i is consistent with the X-ray crystallographic structure of F₁ (Bianchet, et al., 1998). Thus, five of the six molecules of bound P_i were accounted for. Three molecules of bound P_i were at catalytic sites and participated in ATP synthesis or P_i ATP exchange. Two other molecules of bound P_i were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound P_i remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F₁F₀ in DMSO-containing compared with DMSO-free buffers.     

Transport of phosphate from leaves to leguminous root nodules

Summary When P³²-orthophosphate was applied to leaves ofLotus pedunculatus radioactivity was detected in the root nodules. About 50 per cent of the radioactivity was in ATP, ADP and AMP. More than 90 per cent of the P³² compounds were localized in the soluble part of the plant tissue of the nodules. Rhizobial bacteroids contained only 1 per cent of P³² incorporated in the nodules.