Broiler breeder manure phosphorus forms are affected by diet, location, and period of accumulation.

Phosphorus (P) modifications of poultry diets have successfully decreased the total P (TP) in manures, but the effects on manure water-soluble P (WSP(M)) remain unclear. Our objectives were to characterize P forms in broiler breeder manures as affected by dietary P modification, location within the pen, and manure accumulation period. Two diets were formulated with and without phytase to attain 0.40% available P (AvP) during the breeder laying phase (22-64 wk of age). Manure was collected after accumulation periods of 48 h, 3 wk, and 39 wk in locations under the feeder and drinker and under the common area (between the feeder and drinker) of the pen. The TP, WSP(M), orthophosphate, and phytate in manure were measured. Broiler breeders that were fed phytase with a simultaneous reduction in nonphytate P (NPP) produced manures with 15% lower TP than those fed a traditional diet, but did not change WSP(M) when averaged over manure accumulation periods and locations within the pen. Regardless of diet, location within the pen, or accumulation period (r(2) = 0.76), the WSP(M) increased linearly as the manure moisture increased. As manure accumulation periods increased (48 h, 3 wk, and 39 wk), TP manure concentrations increased (11.9, 13.2, and 17.3 g/kg, respectively), orthophosphate proportions increased (73.2, 80.1, and 91.0%, respectively), and phytate proportions decreased (23.1, 17.0, and 6.7%, respectively). The mineralization of phytate and other organic complexes, which drive off carbon dioxide, presumably contributed to the increased orthophosphate and TP concentrations. Keeping breeder manures dry helps to avoid the mineralization of phytate to orthophosphate; this mineralization increased WSP(M) in our study, and thus increased the potential for elevated P loss in runoff when surface applied.

Distribution of ten antibiotic resistance genes in E. coli isolates from swine manure, lagoon effluent and soil collected from a lagoon waste application field.

The prevalence of ten antibiotic resistance genes (ARGs) was evaluated in a total of 616 Escherichia coli isolates from swine manure, swine lagoon effluent, and from soils that received lagoon effluent on a commercial swine farm site in Sampson County, North Carolina (USA). Isolates with ARGs coding for streptomycin/spectinomycin (aadA/strA and strB), tetracycline (tetA and tetB), and sulfonamide (sul1) occurred most frequently (60.6-91.3%). The occurrence of E. coli isolates that carried aadA, tetA, tetB, and tetC genes was significantly more frequent in soil samples (34.0-97.2%) than in isolates from lagoon samples (20.9-90.6%). Furthermore, the frequency of isolates that contain genes coding for aadA and tetB was significantly greater in soil samples (82.6-97.2%) when compared to swine manure (16.8-86.1%). Isolates from the lagoon that carried tetA, tetC, and sul3 genes were significantly more prevalent during spring (63.3-96.7%) than during winter (13.1-67.8%). The prevalence of isolates from the lagoon that possessed the strA, strB, and sul1 resistance genes was significantly more frequent during the summer (90.0-100%) than during spring (66.6-80.0%). The data suggest that conditions in the lagoon, soil, and manure may have an impact on the occurrence of E. coli isolates with specific ARGs. Seasonal variables seem to impact the recovery isolates with ARGs; however, ARG distribution may be associated with mobile genetic elements or a reflection of the initial numbers of resistant isolates shed by the animals.

Magnesium is more efficient than calcium in alleviating aluminum rhizotoxicity in soybean and its ameliorative effect is not explained by the Gouy-Chapman-Stern model.

The mechanistic basis for cation amelioration of Al rhizotoxicity in soybean was investigated through a series of studies comparing protective effects of Ca and Mg against Al inhibition of root elongation in a background 0.8 mM CaSO4 solution (pH 4.3). A modified Gouy-Chapman-Stern model was used to evaluate the effect of cations on electrical potential and Al3+ activity at root plasma membrane surfaces. Activities of Al3+ up to 4.6 microM in the background solution inhibited soybean tap root elongation by more than 80%. There was little or no response in root elongation when Ca and Mg were added to background solutions in the absence of AL: When added to Al-toxic solutions in the micromolar concentration range, Mg was 100-fold more effective than Ca in alleviating Al toxicity, whereas both cations were equally effective when added in the millimolar concentration range. The protective effect of micromolar additions of Mg on root elongation was specific for Al and it failed to alleviate La rhizotoxicity. In contrast to wheat, Mg amelioration of Al toxicity to soybean root elongation at low Mg concentration could not be explained by changes in potential and Al3+ activity at the root plasma membrane surfaces as predicted by a Gouy-Chapman-Stern model. These results suggest that Mg is not acting as an indifferent cation when present at low concentration and implies the involvement of a mechanism other than pure electrostatic effects at the root surface.

Magnesium ameliorates aluminum rhizotoxicity in soybean by increasing citric acid production and exudation by roots.

Superior effectiveness of Mg over Ca in alleviating Al rhizotoxicity cannot be accounted for by predicted changes in plasma membrane Al3+ activity. The influence of Ca and Mg on the production and secretion of citrate and malate, and on Al accumulation by roots was investigated with soybean genotypes Young and PI 416937 which differ in Al tolerance. In the presence of a solution Al3+ activity of 4.6 microM, citrate and malate concentrations of tap root tips of both genotypes increased with additions of either Ca up to 3 mM or Mg up to 50 microM. Citrate efflux rate from roots exposed to Al was only enhanced with Mg additions and exceeded malate efflux rates by as much as 50-fold. Maximum citrate release occurred within 12 h after adding Mg to solution treatments. Adding 50 microM Mg to 0.8 mM CaSO4 solutions containing Al3+ activities up to 4.6 microM increased citrate concentration of tap root tips by 3- to 5-fold and root exudation of citrate by 6- to 9-fold. Plants treated with either 50 microM Mg or 3 mM Ca had similar reductions in Al accumulation at tap root tips, which coincided with the respective ability of these ions to relieve Al rhizotoxicity. Amelioration of Al inhibition of soybean root elongation by low concentrations of Mg in solution involved Mg-stimulated production and efflux of citrate by roots.

nif gene expression in a Nif+, Fix- Bradyrhizobium japonicum variant.

Bradyrhizobium japonicum USDA 110 has been shown to contain several genetically similar, naturally occurring colony morphology variants. One of these variants, L2-110, although devoid of symbiotic nitrogen fixation, retains significant levels of ex planta nitrogen fixation ability relative to other symbiotically competent USDA 110 variants (MN-110 and I-110). Interestingly, Northern blot analyses revealed that L2-110 nodules, despite their lack of symbiotic nitrogen fixation, contained 65% the level of mRNA for dinitrogenase (nif DK) and 64% the level of dinitrogenase reductase (nif H) mRNA relative to MN-110 nodules. Western blot analyses of tissue from the same nodules detected 32% the level of dinitrogenase and 31% the level of dinitrogenase reductase in L2-110 relative to MN-110. L2-110 appears to be a new class of mutant based on the complete absence of symbiotic nitrogen fixation (Fix-) and the presence of significant ex planta nitrogen fixation (Nif+).

Diurnal starch accumulation and utilization in phosphorus-deficient soybean plants.

The effects of phosphorus deficiency on carbohydrate accumulation and utilization in 34-day-old soybean (Glycine max L. Merr.) plants were characterized over a diurnal cycle to evaluate the mechanisms by which phosphorus deficiency restricts plant growth. Phosphorus deficiency decreased the net CO(2) exchange rate throughout the light period. The decrease in the CO(2) exhange rate was associated with a decrease in stomatal conductance and an increase in the internal CO(2) concentration. These observations indicate that phosphorus deficiency increased mesophyll resistance. Assimilate export rate from the youngest fully expanded leaves was decreased by phosphorus deficiency, whereas starch concentrations in these leaves were increased. Higher starch concentrations in phosphorus-deficient youngest fully expanded leaves resulted from a longer period of net starch accumulation and a shorter period of net starch degradation relative to those for phosphorus-sufficient controls. Phosphorus deficiency decreased sucrose-P synthase activity by 27% (averaged over the diurnal cycle), and essentially eliminated diurnal variation in sucrose-P-synthase activity. Diurnal variations in nonstructural carbohydrate concentrations in leaves and stems were also less pronounced in phosphorus-deficient plants than in controls. In phosphorus-deficient plants, only 30% of the whole plant starch present at the end of a light phase was utilized during the subsequent 12-hour dark phase as compared with 68% for phosphorus-sufficient controls. Although phosphorus deficiency decreased the CO(2) exchange rate and whole plant leaf area, accumulation of high starch concentrations in leaves and stems and restricted starch utilization in the dark indicate that growth processes (i.e. sink activities) were restricted to a greater extent than photosynthetic capacity. Further experimentation is required to determine whether decreased starch utilization in phosphorus-deficient plants is the cause or the result of restricted growth.

Energy status and functioning of phosphorus-deficient soybean nodules.

Characterization of the effects of long-term P deficiency and of onset and recovery from P deficiency on bacteroid mass and number per unit nodule mass and energy status of soybean (Glycine max L. Merr.) nodules was used to investigate the mechanisms by which P deficiency decreases symbiotic N(2) fixation. The continuous P deficiency treatment (0.05 millimolar P) significantly decreased the whole plant dry mass, P, and N by 62, 90, and 78%, respectively, relative to the P-sufficient control (1.0 millimolar) at 44 days after transplanting. Specific nitrogenase activity was decreased an average of 28% over a 16-day experimental period by P deficiency. Whole nodules of P-deficient controls contained 70 to 75% lower ATP concentrations than nodules of P-sufficient controls. Energy charge and ATP concentrations in the bacteroid fraction of nodules were not significantly affected by P treatment. However, ATP and total adenylate concentrations and energy charge in the plant cell fraction of nodules were significantly decreased 91, 62, and 50%, respectively, by the P deficiency treatment. Specific nitrogenase activity, energy charge, and ATP concentration in the plant cell fraction increased to the levels of nonstressed controls within 2, 2, and 4 days, respectively, after alleviation of external P limitation, whereas bacteroid mass per unit nodule mass and bacteroid N concentration did not increase to the level of nonstressed controls until 7 days after alleviation of external P limitation. All of these parameters except bacteroid mass per unit nodule mass decreased to the levels of the P-deficient controls by 11 days after onset of external P limitation. Concentration of ATP in the bacteroid fraction was not significantly affected by alteration in the external P supply. Energy charge in the bacteroid fraction from plants recovering from P deficiency was decreased to a small (10%) but significant extent (P < 0.05) at two sampling dates relative to P-sufficient controls. These ATP concentration and energy charge measurements indicate that P deficiency impaired oxidative phosphorylation in the plant cell fraction of nodules to a much greater extent than in the bacteroids. The concurrence of significant changes in specific nitrogenase activity (2 days) and in the energy charge (2 days) and ATP concentration (4 days) in the plant cell fraction during recovery from external P limitation is consistent with the conclusion that P deficiency decreases the specific nitrogenase activity by inhibiting an energy-dependent reaction(s) in the plant cell fraction of the nodules.

Phosphorus stress effects on assimilation of nitrate.

An experiment was conducted to investigate alterations in uptake and assimilation of NO(3) (-) by phosphorus-stressed plants. Young tobacco plants (Nicotiana tabacum [L.], cv NC 2326) growing in solution culture were deprived of an external phosphorus (P) supply for 12 days. On selected days, plants were exposed to (15)NO(3) (-) during the 12 hour light period to determine changes in NO(3) (-) assimilation as the P deficiency progressed. Decreased whole-plant growth was evident after 3 days of P deprivation and became more pronounced with time, but root growth was unaffected until after day 6. Uptake of (15)NO(3) (-) per gram root dry weight and translocation of absorbed (15)NO(3) (-) out of the root were noticeably restricted in -P plants by day 3, and effects on both increased in severity with time. Whole-plant reduction of (15)NO(3) (-) and (15)N incorporation into insoluble reduced-N in the shoot decreased after day 3. Although the P limitation was associated with a substantial accumulation of amino acids in the shoot, there was no indication of excessive accumulation of soluble reduced-(15)N in the shoot during the 12 hour (15)NO(3) (-) exposure periods. The results indicate that alterations in NO(3) (-) transport processes in the root system are the primary initial responses limiting synthesis of shoot protein in P-stressed plants. Elevated amino acid levels evidently are associated with enhanced degradation of protein rather than inhibition of concurrent protein synthesis.

Effects of Bradyrhizobium japonicum and Soybean (Glycine max (L.) Merr.) Phosphorus Nutrition on Nodulation and Dinitrogen Fixation.

Cells of Bradyrhizobium japonicum were grown in media containing either 1.0 mM or 0.5 muM phosphorus. In growth pouch experiments, infection of the primary root of soybean (Glycine max (L.) Merr.) by B. japonicum USDA 31, 110, and 142 was significantly delayed when P-limited cells were applied to the root. In a greenhouse experiment, B. japonicum USDA 31, 110, 122, and 142 grown with sufficient and limiting P were used to inoculate soybeans which were grown with either 5 muM or 1 mM P nutrient solution. P-limited cells of USDA 31 and 110 formed significantly fewer nodules than did P-sufficient cells, but P-limited cells of USDA 122 and 142 formed more nodules than P-sufficient cells. The increase in nodule number by P-limited cells of USDA 142 resulted in significant increases in both nodule mass and shoot total N. In plants grown with 1 mM P, inoculation with P-limited cells of USDA 110 resulted in lower total and specific nitrogenase activities than did inoculation with P-sufficient cells. Nodule numbers, shoot dry weights, and total N and P were all higher in plants grown with 1 mM P, and plants inoculated with USDA 31 grew poorly relative to plants receiving strains USDA 110, 122, and 142. Although the effects of soybean P nutrition were more obvious than those of B. japonicum P nutrition, we feel that it is important to develop an awareness of the behavior of the bacterial symbiont under conditions of nutrient limitation similar to those found in many soils.

Investigation of the role of phosphorus in symbiotic dinitrogen fixation.

The interactive effects of phosphorus supply and combined nitrogen (nitrate) on dry matter and nitrogen accumulation by nodulated soybean (Glycine max L. Merr.) plants, and the relative effects of phosphorus supply on nodule number, mass, and function in comparison to host plant growth were used to investigate the role of phosphorus in symbiotic dinitrogen fixation. Mixed positive and negative phosphorus by nitrogen source interactions indicated that severe phosphorus deficiency markedly impaired both host plant growth and symbiotic dinitrogen fixation and that symbiotic dinitrogen fixation has a higher phosphorus requirement for optimal functioning than either host plant growth or nitrate assimilation. In the whole plant phosphorus concentration range of 0.8 to 1.5 grams per kilogram dry weight, plants supplied with 20 millimolar nitrate accumulated significantly more dry matter and nitrogen than symbiotic plants without nitrate. This suggested that the higher phosphorus requirement for symbiotic dinitrogen fixation is internal rather than being associated with differences in the ability of roots in the two nitrogen regimes to absorb phosphorus from the external solution. Increasing the phosphorus concentration in plants solely dependent on dinitrogen fixation resulted in highly significant (P = 0.0001) increases in whole plant nitrogen concentration as well as highly significant increases (P = 0.0001) in whole plant dry matter and nitrogen accumulation. This indicated a greater responsiveness of symbiotic dinitrogen fixation than of host plant growth to improvement in phosphorus nutrition. The large increases in whole plant nitrogen concentration were associated with about 3.5-fold increases in the ratio of nodule mass to whole plant mass and about 2-fold increases in specific acetylene reduction (nitrogenase) activity of the nodules. The large increase in nodule mass (>30-fold) between the 0 and 2.0 millimolar phosphorus levels resulted from 11- and 3-fold increases in nodule number per plant and average mass of individual nodules, respectively. Root mass per plant over the same concentration range increased 3.5-fold. These results indicate that phosphorus has specific roles in nodule initiation, growth, and functioning in addition to its involvement in host plant growth processes.

Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative.

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N(2)-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K(2)HPO(4)-0.08% MgSO(4) . 7H(2)O-0.02% NaCl-0.001% FeCl(3) . 6H(2)O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.

Characterization of a Mannitol-Utilizing, Nitrogen-Fixing Bradyrhizobium japonicum USDA 110 Derivative.

We have isolated a colonial derivative of Bradyrhizobium japonicum USDA 110 (designated MN-110) that is both mannitol utilizing and N(2) fixing. Derivative MN-110 showed growth on mannitol and glucose similar to that of non-N(2)-fixing, mannitol-utilizing L2-110. Derivative MN-110 showed high constitutive and induced d-mannitol dehydrogenase activity (similar to L2-110) relative to N(2)-fixing, non-mannitol-utilizing I-110. Hybridization to EcoRI and HindIII total DNA digests with cloned USDA 110 nif DK and nif H genes revealed similar patterns for non-N(2)-fixing mannitol-utilizing derivative L1-110 and derivative MN-110. Symbiotic tests with soybean cultivars Ransom and Lee indicate MN-110 to be a superior N(2)-fixing derivative compared with derivative I-110 and the parent strain USDA 110. However, these differences were not revealed when comparing 28-day-old soybean-B. japonicum associations but were apparent in 49-day-old associations. It was apparent from this work that mannitol utilization was not necessarily correlated to symbiotic effectiveness in B. japonicum and that gene rearrangements were not responsible for differences in N(2) fixation between L1-110 or L2-110 and MN-110.

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars.

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N(2) than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N(2) than did the USDA 110 during the 20-day period. The superior N(2) fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N(2)-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N(2)-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N(2) in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N(2)-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N(2) fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N(2)-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.

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 (ms(1)) and nodulation (rj(1)), 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 KNO(3). 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 (N(2)-fixing plants) to roots and leaves (NO(3)-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 N(2) 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 N(2)-fixing plants (63%). Hence, vegetative-tissue nitrogen mobilization to reproductive tissue was a more prominent process in N(2)-fixing than in nitrate-dependent plants. N(2)-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).

Assimilation of NO(3) Taken Up by Plants in the Light and in the Dark.

An experiment was conducted to determine the extent that NO(3) (-) taken up in the dark was assimilated and utilized differently by plants than NO(3) (-) taken up in the light. Vegetative, nonnodulated soybean plants (Glycine max L. Merrill, ;Ransom') were exposed to (15)NO(3) (-) throughout light (9 hours) or dark (15 hours) phases of the photoperiod and then returned to solutions containing (14)NO(3) (-), with plants sampled subsequently at each light/dark transition over 3 days. The rates of (15)NO(3) (-) absorption were nearly equal in the light and dark (8.42 and 7.93 micromoles per hour, respectively); however, the whole-plant rate of (15)NO(3) (-) reduction during the dark uptake period (2.58 micromoles per hour) was 46% of that in the light (5.63 micromoles per hour). The lower rate of reduction in the dark was associated with both substantial retention of absorbed (15)NO(3) (-) in roots and decreased efficiency of reduction of (15)NO(3) (-) in the shoot. The rate of incorporation of (15)N into the insoluble reduced-N fraction of roots in darkness (1.10 micromoles per hour) was somewhat greater than that in the light (0.92 micromoles per hour), despite the lower rate of whole-plant (15)NO(3) (-) reduction in darkness.A large portion of the (15)NO(3) (-) retained in the root in darkness was translocated and incorporated into insoluble reduced-N in the shoot in the following light period, at a rate which was similar to the rate of whole-plant reduction of (15)NO(3) (-) acquired during the light period. Taking into account reduction of NO(3) (-) from all endogenous pools, it was apparent that plant reduction in a given light period ( approximately 13.21 micromoles per hour) exceeded considerably the rate of acquisition of exogenous NO(3) (-) (8.42 micromoles per hour) during that period. The primary source of substrate for NO(3) (-) reduction in the dark was exogenous NO(3) (-) being concurrently absorbed. In general, these data support the view that a relatively small portion (<20%) of the whole-plant reduction of NO(3) (-) in the light occurred in the root system.

Effect of N-source on soybean leaf sucrose phosphate synthase, starch formation, and whole plant growth.

Soybeans (Glycine max L. Merr. cv Tracy and Ransom) were grown under N(2)-dependent or NO(3) (-)-supplied conditions, and the partitioning of photosynthate and dry matter was characterized. Although no treatment effects on photosynthetic rates were observed, NO(3) (-)-supplied plants in both cultivars had lower starch accumulation rates than N(2)-dependent plants. Leaf extracts of NO(3) (-)-supplied plants had higher activities of sucrose phosphate synthase (SPS) and cytoplasmic fructose-1,6-bisphosphatase (FBPase) than N(2)-dependent plants. The variation in starch accumulation was correlated negatively with the activity of SPS, but not the activity of FBPase, UDP-glucose pyrophosphorylase, or ADP-glucose pyrophosphorylase. These results suggested that starch accumulation is biochemically controlled, in part, by the activity of SPS. Leaf starch content at the beginning of the photoperiod was lower in NO(3) (-)-supplied plants than N(2)-dependent plants in both cultivars which suggested that net starch utilization as well as accumulation was affected by N source.Total dry matter accumulation and dry matter distribution was affected by N source in both cultivars, but the cultivars differed in how dry matter was partitioned between the shoot and root as well as within the shoot. The activity of SPS was correlated positively with total dry matter accumulation which suggested that SPS activity is related to plant growth rate. The results suggested that photosynthate partitioning is an important but not an exclusive factor which determines whole plant dry matter distribution.

Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves.

The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by (14)CO(2) assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total (14)CO(2) fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = -0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.Four nodulated soybean cultivars were grown to maturity in a greenhouse. Fully expanded leaves at the top of the canopy were sampled during vegetative growth (45 days), at flowering (79 days), and at mid-podfill (120 days). In general, activities of SPS and uridine-5'-diphosphatase were highest during vegetative growth, and they decreased during reproductive development, whereas activity of sucrose synthase and leaf starch content tended to increase. Leaf starch was negatively correlated with levels of SPS (r = -0.71). The results support the postulate that sucrose-P synthetase is a key control point regulating the photosynthetic formation of sucrose, and, hence, starch.

Relative Content of NO(3) and Reduced N in Xylem Exudate as an Indicator of Root Reduction of Concurrently Absorbed NO(3).

It is unclear if the relative content of NO(3) (-) and reduced N in xylem exudate provides an accurate estimate of the percentage reduction of concurrently absorbed NO(3) (-) in the root. Experiments were conducted to determine whether NO(3) (-) and reduced N in xylem exudate of vegetative, nonnodulated soybean plants (Glycine max [L.] Merr., ;Ransom') originated from exogenous recently absorbed (15)NO(3) (-) or from endogenous (14)N pools. Plants either were decapitated and exposed to (15)NO(3) (-) solutions for 2 hours or were decapitated for the final 20 minutes of a 50-minute exposure to (15)NO(3) (-) in the dark and in the light. Considerable amounts of (14)NO(3) (-) and reduced (14)N were transported into the xylem, but almost all of the (15)N was present as (15)NO(3) (-). Dissimilar changes in transport of (14)NO(3) (-), reduced (14)N and (15)NO(3) (-) during the 2 hours of sap collection resulted in large variability over time in the percentage of total N in the exudate which was reduced N. Over a 20-minute period the rate of (15)N transport into the xylem of decapitated plants was only 21 to 36% of the (15)N delivered to the shoot of intact plants. Based on the proportion of total (15)N which was found as reduced (15)N in exudate and in intact plants in the dark, it was estimated that 5 to 17% of concurrently absorbed (15)NO(3) (-) was reduced in the root. This was much less than the 38 to 59% which would have been predicted from the relative content of total NO(3) (-) and total reduced N in the xylem exudate.

Ion balance, uptake, and transport processes in n(2)-fixing and nitrate- and urea-dependent soybean plants.

The objective of this study was to examine the influence of N(2) fixation and NO(3) (-)-N and urea-N assimilation on ion balance, uptake, and transport processes in soybean (Glycine max L. Merr.).Inoculated plants were grown in Perlite supplied daily with nutrient solutions which contained zero-N, 10 and 20 millimolar NO(3) (-)-N, and 10 and 20 millimolar urea-N, and they were sampled 41, 76, and 151 days after transplanting. Total uptake of inorganic cations and anions was determined by analysis of tissue for K(+), Ca(2+), Mg(2+), Na(+), total N from NO(3) (-), total S, H(2)PO(4) (-), and Cl(-). Differences in total inorganic cations (C) and inorganic anions (A) in plant tissue were used to estimate total carboxylate content.Internal OH(-) generation resulting from excess cation uptake (net H(+) excretion) by the roots accounted for more than 89% of the carboxylate accumulation in N(2)- and urea-fed plants, while OH(-) generation resulting from SO(4) (2-) reduction accounted for less than 11%. Shoots contained over 89% of the total plant carboxylate content. Malate balanced about 75% of the excess inorganic cationic charge of the xylem sap; allantoate and aspartate balanced most of the remaining charge. These results indicate that carboxylates (primarily malate) are synthesized in roots of N(2)- and urea-fed plants and transported to the shoots in the xylem to maintain charge balance. The high malate concentration resulted in the C/N weight ratio of xylem sap from N(2)-fed plants being >2.0, even though 83% of the N was transported as allantoin and allantoic acid which have a C/N ratio of 1.0. The data emphasize that C and N content of N compounds should not be the sole basis for calculating the C/N weight ratio of xylem sap.The C-to-A uptake ratio for plants supplied 10 millimolar NO(3) (-) ranged from 1.24 to 1.57 during development, indicating that internal OH(-) was generated both by excess cation uptake and by NO(3) (-) and SO(4) (2-) reduction. The C-to-A uptake ratio for 20 millimolar NO(3) (-) -fed plants ranged from 0.86 to 0.96 during development, indicating a small net OH(-) efflux from the roots for support of excess anion uptake. On a seasonal basis, only 15% of the OH(-) generated during NO(3) (-) and SO(4) (2-) reduction was associated with OH(-) efflux (excess anion uptake), while 85% was associated with carboxylate accumulation. The malate concentration in xylem sap from plants supplied 20 millimolar NO(3) (-) was only one-third that of N(2)- and urea-fed plants; however, it did balance 75% of the excess inorganic cationic charge. Potassium, recycling to accommodate excess anion uptake by 20 millimolar NO(3)-fed plants, was calculated to involve at most 17% of the total K(+) absorbed during the 41- to 76-day growth interval.

Evaluation of the Relative Ureide Content of Xylem Sap as an Indicator of N(2) Fixation in Soybeans: GREENHOUSE STUDIES.

The use of the relative ureide content of xylem sap [(ureide-N/total N) x 100] as an indicator of N(2) fixation in soybeans (Merr.) was examined under greenhouse conditions. Acetylene treatments to inhibit N(2) fixation were imposed upon the root systems of plants totally dependent upon N(2) fixation as their source of N and of plants dependent upon both N(2) fixation and uptake of exogenous nitrate. Significant decreases in the total N concentration of xylem sap from plants of the former type were observed, but no significant decrease was observed in the total N concentration of sap from the latter type of plants. In both types of plants, acetylene treatment caused significant decreases in the relative ureide content of xylem sap. The results provided further support for a link between the presence of ureides in the xylem and the occurrence of N(2) fixation in soybeans. The relative ureide content of xylem sap from plants totally dependent upon N(2) fixation was shown to be insensitive to changes in the exudation rate and total N concentration of xylem sap brought about by diurnal changes in environmental factors. There was little evidence of soybean cultivars or nodulating strains affecting the relative ureide content of xylem sap. ;Ransom' soybeans nodulated with Rhizobium japonicum strain USDA 110 were grown under conditions to obtain plants exhibiting a wide range of dependency upon N(2) fixation. The relative ureide content of xylem sap was shown to indicate reliably the N(2) fixation of these plants during vegetative growth using a (15)N method to measure N(2) fixation activity. The use of the relative ureide content of xylem sap for quantification of N(2) fixation in soybeans should be evaluated further.