Insufficient nitrogen supply from symbiotic fixation reduces seasonal crop growth and nitrogen mobilization to seed in highly productive soybean crops

Nitrogen (N) supply can limit the yields of soybean [Glycine max (L.) Merr.] in highly productive environments. To explore the physiological mechanisms underlying this limitation, seasonal changes in N dynamics, aboveground dry matter (ADM) accumulation, leaf area index (LAI) and fraction of absorbed radiation (fAPAR) were compared in crops relying only on biological N₂ fixation and available soil N (zero-N treatment) versus crops receiving N fertilizer (full-N treatment). Experiments were conducted in seven high-yield environments without water limitation, where crops received optimal management. In the zero-N treatment, biological N₂ fixation was not sufficient to meet the N demand of the growing crop from early in the season up to beginning of seed filling. As a result, crop LAI, growth, N accumulation, radiation-use efficiency and fAPAR were consistently higher in the full-N than in the zero-N treatment, leading to improved seed set and yield. Similarly, plants in the full-N treatment had heavier seeds with higher N concentration because of greater N mobilization from vegetative organs to seeds. Future yield gains in high-yield soybean production systems will require an increase in biological N₂ fixation, greater supply of N from soil or fertilizer, or alleviation of the trade-off between these two sources of N in order to meet the plant demand.     

Effect of N source during soybean pod filling on nitrogen and sulfur assimilation and remobilization

During pod filling, a grain legume remobilizes vegetative nitrogen and sulfur to its developing fruit. This study was conducted to determine whether different nitrogen sources affected N and S assimilation and remobilization during pod filling. Well-nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea assimilated 0%, 37%, or 114% more N, respectively, and 25%, 46%, or 56% more S, respectively, than did the average non-nodulated control plant fed 5.0 mM KNO₃. Thus, N source during pod filling greatly affected both N and S assimilation. Depending upon N source, plant N concentration during pod filling decreased from 2.96% to between 1.36% and 1.82%. Non-nodulated control plants fed 5.0 mM KNO₃ had the highest residual N at harvest. During the same treatments, plant S concentration decreased from 0.246% to a relatively uniform 0.215%. Thus, during pod filling, vegetative N was seemingly remobilized more efficiently (38–54%) than was S (13%). N source also affected seed yield and seed quality. Non-nodulated control plants fed 5.0 mM KNO₃ produced the lowest yield (21.1 g seeds plant^-1), whereas well nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea produced yields of 26.2 g, 31.8 g, and 36.7 g seeds plant^-1, respectively. Non-nodulated plants fed 2.5 mM urea yielded 28.6 g of seeds plant^-1. Seed N concentrations of non-nodulated plants and nodulated plants fed 2.5 mM urea were high, 6.30% and 6.11% N, respectively, whereas their seed S concentrations were low, 0.348% and 0.330% S, respectively. N sources that produced both a relatively high seed yield and seed N concentration (i.e., a relatively high total seed N plant^-1) produced a proportionately smaller increase in total seed sulfur. Consequently, seed quality, as judged solely by seed S concentration, was lowered.     

Growth,nodulation and nitrogen fixation in soybean as affected by air humidity and root temperature

Growth, nodulation and N₂ fixation inGlycine max L. Merr., cv. Biison as affected by the relative humidity of air (RH) during the dark period (95 or 50 – 65 %) and day/night root temperature (T_r) (28/28, 25/25, 18/18, 22/28, 22/18 °C) were studied. The growth parameters (plant fresh and dry mass, yield), nodulation (nodule number and fresh mass) and N₂ fixation abilities (total nitrogen content, nitrogenase activity) increased significantly with the increasing T_r. In addition, at the same T_r during the day all studied parameters were increased at the higher T_r during the dark period. Growth, nodulation and N₂ fixation were significantly enhanced at low RH. The findings indicate that all studied parameters could be regulated by environmental factors during the dark period.     

Regulation of soybean nodule phosphoenolpyruvate carboxylase in vivo

The sensitivity of soybean ( Glycine max L. Merr, cv. PS47) nodule phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) to inhibition by L-malate in vitro increased when well-nodulated plants were subjected to decapitation (shoot removal). There was no effect of decapitation on the apparent K_m of the enzyme for its substrate PEP but the I₅₀ (L-malate) decreased from 4.2 to 1.7 m M. The total amount of PEP doubled and that of malate decreased by half in the nodules of decapitated plants relative to the control plants. This observation was consistent with a decrease in the activity of PEPC in vivo as a result of the increased malate sensitivity of the enzyme observed in vitro. Sucrose levels in the nodules declined in response to decapitation but there were no effects on the levels of glucose, fructose, pyruvate, 2-oxoglutarate, glutamine or glutamate. The results are discussed in terms of the role of protein phosphorylation in the regulation of PEPC activity in legume nodules.     

Ineffective utilization of nitrate by soybean during pod fill

The relative effectiveness of nitrate, allantoin, or nitrate plus allantoin as sources of nitrogen for the indeterminate soybean plant [ Glycine max (L.) Merr cv. Harper] was studied throughout vegetative and reproductive growth. All plants were provided with 3.0 m M nitrogen and were grown hydroponically in growth chambers. During vegetative and early reproductive growth, plants given nitrate or nitrate plus allantoin grew faster than plants provided allantoin only. However, during pod fill, plants provided with allantoin or allantoin plus nitrate gained weight more rapidly than plants receiving just nitrate. More importantly, at maturity plants that had been provided with allantoin or allantoin plus nitrate during pod fill were 30% heavier in total dry weight, 50% higher in nitrogen content, and 50% higher in seed yield than plants that had received just nitrate. At full bloom, all plants were inoculated with the same culture of Bradyrhizobium japonicum , and twice each week throughout pod fill each plant was assayed for nitrogen fixation (acetylene reduction). Correlation coefficients obtained by linear regression analysis show a strong positive correlation between the measured rate of nitrogen fixation and maximum plant fresh weight (r = 0.83), total plant nitrogen (r = 0.81), or seed yield (r = 0.76). The fact that nitrogen fixation during pod fill stimulates plant growth and seed yield, coupled with the facts that nitrate blocks nodulation and is not used efficiently during pod fill by the soybean plant, may explain why seed yield of field-grown soybeans usually does not respond to added fertilizer nitrogen. Thus, it is suggested that enhanced nitrogen fixation may be the key factor in improving soybean seed yield.     

Application of genistein to inocula and soil to overcome low spring soil temperature inhibition of soybean nodulation and nitrogen fixation

In the soybean (Glycine max. (L.) Merr)– Bradyrhizobium japonicum symbiosis, suboptimal root zone temperatures (RZTs) slow nodule development by disruption of the interorganismal signal exchange between the host plant and bradyrhizobia. Two field experiments were conducted on two adjacent sites in 1994 to determine whether the incubation of B. japonicum with genistein prior to application as an inoculant, or genistein, without B. japonicum, applied onto seeds in the furrow at the time of planting, increased soybean nodulation, N fixation, and total N yield. The results of these experiments indicated that genistein application increased nodule number and nodule dry matter per plant and hastened the onset of N fixation during the early portion of the soybean growing season, when the soils were still cool. Because these variables were improved, total fixed. N, fixed N as a percentage of total plant N, and N yield increased due to genistein application. The interaction between genistein application and soybean cultivars indicated that genistein application was more effective on N-stressed plants.     

Vascular transport and soybean nodule function: II. A role for phloem supply in product export*

Abstract The ureide content of soybean (Glycine max (L.) Merr.) nodules was unaffected by variations in the transpirational rate, while whole plant manipulations designed to decrease phloem supply to nodules resulted in lower rates of nitrogenase activity and an increase in the ureide content of the nodules. The rate of ureide export from the nodule was estimated from the exponential rate of decrease in the pool size of ureides in nodules, following exposure to an N₂-free atmosphere (Ar:O₂). Export was greatly reduced under treatments which reduced phloem supply to the nodule. A water budget for nodules suggested that the delivery of water to the nodule via mass flow in the phloem was comparable to that required for export of ureides from the nodule in the xylem from the nodule. Therefore, we suggest that xylem export from nodules is related to the phloem supply to the nodule rather than to the transpirational flux in the parent root. This suggestion is related to the reported decreases in nodule permeability to gases under conditions of phloem deprivation.     

Effect of Rhizobium sp. BARIRGm901 inoculation on nodulation,nitrogen fixation and yield of soybean (Glycine max) genotypes in gray terrace soil

Soybean plants require high amounts of nitrogen, which are mainly obtained from biological nitrogen fixation. A field experiment was conducted by soybean (Glycine max) genotypes, growing two varieties (Shohag and BARI Soybean6) and two advanced lines (MTD10 and BGM02026) of soybean with or without Rhizobium sp. BARIRGm901 inoculation. Soybean plants of all genotypes inoculated with Rhizobium sp. BARIRGm901 produced greater nodule numbers, nodule weight, shoot and root biomass, and plant height than non-inoculated plants. Similarly, inoculated plants showed enhanced activity of nitrogenase (NA) enzyme, contributing to higher nitrogen fixation and assimilation, compared to non-inoculated soybean plants in both years. Plants inoculated with Rhizobium sp. BARIRGm901 also showed higher pod, stover, and seed yield than non-inoculated plants. Therefore, Rhizobium sp. BARIRGm901 established an effective symbiotic relationship with a range of soybean genotypes and thus increased the nodulation, growth, and yield of soybean grown in gray terrace soils in Bangladesh.     

Vascular transport and soybean nodule function: nodule xylem is a blind alley, not a throughway

Abstract. The only published consideration of product removal from the soybean root nodule hypothesizes that the peripheral xylem circuit of this determinate nodule structure is flushed by the transpiration stream. However, dyes fed to the transpiration stream through a cut root distal to the nodule do not enter the nodule, and the observed movement of radio-tracers from the root into the nodule can be explained by simple diffusion, Also, there are few xylem elements in the nodule, and these elements are of a small diameter, such that this path can not act as a functional loop of the root system. Further, in this study, nodule vascular strands were never observed to be continuous about the nodule, but were observed to end at the nodule tip in a loop within an intact, closed endodermal sac. Nodule vascular tissue was invested in a pericycle of at least three cell layers. These cells are suggested to be active in the loading of the xylem apoplast with ureides, such that the xylem of the nodule always operates in an export role. Nodule water requirements may be supplied via the phloem or the root cortex apoplasm.     

Structural changes in the innercortex cells of soybean root nodules are induced by short-term exposure to high salt or oxygen concentrations

After a 2 h exposure of intact soybean nodules to high concentrations of NaCl (100mol m_?3) or oxygen (8OkPa O₂), morphometric computations carried out using an image analysis technique on semi-thin sections showed that both treatments induced a decrease in the area of the inner-cortex cells, which were then characterized by a tangential elongation. In contrast, no significant change in area occurred in the middle-cortex cells although their elongation decreased. Electron microscopic observations showed that in the inner-cortex cells changes included the presence of wall infoldings, an enlarged periplasmic space and a lobate nucleus whose chromatin distribution differed from that of the control. Structural changes also occurred in the endoplasmic reticulum, microbodies, mitochondria and plastids. From several of these changes, which are similar to those noted in osmocontractil cells in response to external stimuli, it can be hypothesized that the inner cortex may provide a potential mechanism for the control of oxygen diffusion through the nodules.     

Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean

The effects of increasing rhizosphere pO₂on nitrogenase activity and nodule resistance to O₂diffusion were investigated in soybean plants [Glycine max (L.) Merr. cv. Harosoy 63] in which nitrogenase (EC 1.7.99.2) activities were inhibited by (a) removal of the phloem tissue at the base of the stem (stem girdling), (b) exposure of roots to 10 mM NO₃over 5 days (NO₃-treated), or (c) partial inactivation of nitrogenase activity by an exposure of nodulated roots to 100 kPa O₂(O₂-inhibitcd). In control plants and in plants which had been treated with 100 kPa O₂, increasing rhizosphere O₂concentrations in 10 kPa increments from 20 to 70 kPa did not alter the steady-state nitrogenase activity. In contrast, in plants in which nitrogenase activities were depressed by stem girdling or by exposure to NO₃, increasing rhizosphere pO₂resulted in a recovery of 57 or 67%, respectively, of the initial, depressed rates of nitrogenase activity. This suggests that the nitrogenase activity of stem-girdled and NO₃-treated soybeans was O₂-limited. For each treatment, theoretical resistance values for O₂diffusion into nodules were estimated from measured rates of CO₂exchange, assuming a respiratory quotient of 1.1 and 0 kPa of O₂in the infected cells. At an external partial pressure of 20 kPa O₂, the stem-girdled and NO₃^--treated plants displayed resistance values which were 4 to 8.6 times higher than those in the nodules of the control plants. In control and O₂-inhibited plants, increases in pO₂from 20 to 70 kPa in 10 kPa increments resulted in a 2.5- to 3.9-fold increase in diffusion resistance to O₂, and had little effect on either respiration or nitrogenase activity. In contrast, in stem-girdled and NO₃^--treated plants, increases in external pO₂had little effect on diffusion resistance to O₂, but resulted in a 2.3- to 3.2-fold increase in nodule respiration and nitrogenase activity. These results are consistent with stem-girdling and NO₃^--inhibition treatments limiting phloem supply to nodules causing an increase in diffusion resistance to O₂at 20 kPa and an apparent insensitivity of diffusion resistance to increases in external pO₂.     

Vascular transport and soybean nodule function. III: Implications of a continual phloem supply of carbon and water

Abstract. In soybean, stores of carbon within the leaf have been demonstrated to support nodule metabolism under both photosynthetic and non-photosynthetic conditions. Indeed, a net depletion of nodule starch is observed only under conditions of suboptimal rates of nodule metabolism. Therefore, maximal rates of nodule metabolism are associated with a continual supply of phloem sap to the nodule, delivering water, carbon and other solutes. A restriction of phloem supply to the nodule may result in changes in turgor between the apoplast of the export pathway and the symplast of the nodule. This change may cause the observed decrease in the permeability to gases and to the rate of product export from nodules deprived of a phloem supply. It is suggested that nodule metabolism is homeostatically regulated in terms of internal O₂ levels by the delivery of phloem water and solutes.     

Peroxisomes of soybean (Glycine max) root nodule vascular parenchyma cells contain a "nodule—specific" urate oxidase

The cortex of soybean ( Glycine max L. cv. Centennial) nodules contain an organellerich layer of vascular parenchyma tissue, which encircles the elaborate vascular tissue of the nodule. Peroxisomes with small, electron-opaque nucleoids are found in the vascular parenchyma cells. Positive cytochemical staining for catalase (EC 1.11.1.6) confirms their morphological identification as peroxisomes. Activities of both glycolate oxidase (EC 1.1.3.1) and urate oxidase (EC 1.7.3.3) were detected cytochemically in these peroxisomes. Nodule-specific urate oxidase was localized principally in the nucleoid region of these vascular parenchyma peroxisomes, as indicated by immunogold labelling using antibodies against nodulin-35, the nodule-specific urate oxidase. The density of urate oxidase immunogold labelling in the vascular parenchyma peroxisome nucleoid is similar to that of the more well-characterized interstitial cell peroxisomes of the infected zone. These results show that the induction of nodule-specific urate oxidase may be induced in tissue outside of the infected zone.     

Aerenchyma formation and recovery from hypoxia of the flooded root system of nodulated soybean

BACKGROUND AND AIMS: Flooding results in hypoxia of the root system to which N2 fixation of nodulated roots can be especially sensitive. Morphological adaptions, such as aerenchyma formation, can facilitate the diffusion of oxygen to the hypoxic tissues. Using soybean, the aim of the study was to characterize the morphological response of the nodulated root system to flooding and obtain evidence for the recovery of N metabolism. METHODS: Sections from submerged tissues were observed by light microscopy, while sap bleeding from the xylem was analysed for nitrogenous components. KEY RESULTS: Flooding resulted in the rapid formation of adventitious roots and aerenchyma between the stem (immediately above the water line), roots and nodules. In the submerged stem, taproot, lateral roots and adventitious roots, lysigenous aerenchyma arose initially in the cortex and was gradually substituted by secondary aerenchyma arising from cells derived from the pericycle. Nodules developed aerenchyma from cells originating in the phellogen but nodules situated at depths greater than 7-8 cm showed little or no aerenchyma formation. As a result of aerenchyma formation, porosity of the taproot increased substantially between the 4th and 7th days of flooding, coinciding with the recovery of certain nitrogenous products of N metabolism of roots and nodules transported in the xylem. Thus, on the first day of flooding there was a sharp decline in xylem ureides and glutamine (products of N2 fixation), together with a sharp rise in alanine (product of anaerobic metabolism). Between days 7 and 10, recovery of ureides and glutamine to near initial levels was recorded while recovery of alanine was partial. CONCLUSIONS: N metabolism of the nodulated soybean root system can recover at least partially during a prolonged period of flooding, a process associated with aerenchyma formation.     

Soybean nodule gas permeability,nitrogen fixation and dirunal cycles in soil temperature

While diurnal cycles in nitrogen fixation rates are sometimes assumed to result from diurnal variation in photosynthetically active radiation, contradicting evidence exists that indicate soil temperature is the primary environmental influence. These studies assessed the significance of temperature on soybean nitrogen fixation under field conditions. Two groups of intact field-grown soybean plants, one at ambient and the other exposed to a 10°C diurnal variation in soil temperature, were nondestructively assayed for acetylene reduction rates. Activity was closely associated with soil temperature (R²=0.85), even when temperature was 12 h out of phase with ambient. Data were also obtained to determine if the effects of rhizosphere temperature on nitrogen fixation are mediated through an effect on the nodule oxygen permeability. Nodule oxygen permeability of intact, aeroponically grown soybean was closely correlated with the diurnal changes in temperature (R²=0.90).     

A non-destructive field assay for soybean nitrogen fixation by acetylene reduction

Summary A system for employing open-ended root chambers to measurein situ acetylene reduction rates under field conditions is described. Gas mixtures containing about 2 mbar acetylene were continuously flowed through the chambers providing a continuous record of acetylene reduction. These chambers have been used to measure acetylene reduction rates of soybeans during three growing seasons. The system has proved to be reliable with a high degree of precision. The large amount of plant-to-plant variability observed in N₂ fixation research has been confirmed by the data collected with this system. However, such variability in physiological studies can be reduced by using a non-destructive system to compare the response of an individual plant with its rates before treatment.     

A rapid non-destructive assay to quantify soybean nodule gas permeability

The low gas permeability of a diffusion barrier in the cortex of soybean nodules plays a significant role in the protection of nitrogenase from oxygen inactivation. It may also set an upper limit on nodule respiration and nitrogen fixation rates. Two methods which have been used to quantify the gas permeability of leguminous nodules are reviewed and found to be unreliable. A new assay technique for determining both the nodule activity and gas permeability is developed and tested. This ‘lag-phase’ assay is based on the time nodules require to reach steady-state ethylene production after being exposed to acetylene. The technique is rapid, insensitive to errors in biochemical parameters associated with nitrogenase, and is non-destructive. The method was tested with intact aeroponically grown soybean plants for which the mean nodule gas permeability was found to be 13.3×10⁻³ mms⁻¹. This corresponds to a layer of cells approximately 35 um thick and is consistent with previously reported values.     

Potential for increasing biological nitrogen fixation in soybean

The importance of soybean as a source of oil and protein, and its ability to grow symbiotically on low-N soils, point to its continued status as the most valuable grain legume in the world. With limited new land on which to expand, and emphasis on sustainable systems, increases in soybean production will come mostly from increased yield per unit area. Improvements in biological nitrogen fixation can help achieve increased soybean production, and this chapter discusses research and production strategies for such improvement.The soybean-Bradyrhizobium symbiosis can fix about 300 kg N ha^-1 under good conditions. The factors which control the amount of N fixed include available soil N, genetic determinants of compatibility in both symbiotic partners and lack of other yield-limiting factors. Response to inoculation is controlled by the level of indigenous, competing bradyrhizobia, the N demand and yield potential of the host, and N availability in the soil.Research efforts to improve BNF are being applied to both microbe and soybean. While selection continues for effective, naturally occurring bradyrhizobia for inoculants and the use of improved inoculation techniques, genetic research on bradyrhizobia to improve effectiveness and competitiveness is advancing. Selection, mutagenesis and breeding of the host have focused on supernodulation, restricted nodulation of indigenous B. japonicum, and promiscuous nodulation with strains of bradyrhizobia from the cowpea cross-inoculation group. The research from the host side appears closer to being ready for practical use in the field.Existing knowledge and technology still has much to offer in improving biological nitrogen fixation in soybean. The use of high-quality inoculants, and education about their benefits and use can still make a significant contribution in many countries. The importance of using the best adapted soybean genotype with a fully compatible inoculant cannot be overlooked, and we need to address other crop management factors which influence yield potential and N demand, indirectly influencing nitrogen fixation. The implementation of proven approaches for improving nitrogen fixation in existing soybean production demands equal attention as received by research endeavours to make future improvements.