Abstract Symbiotic N2 fixation research thus far has been primarily focused on selection of bacteria. However, little progress in impacting crop yields has resulted from this approach. Bacteria introduced in field soils rarely compete well with indigenous bacteria, including mutated lines selected for high nitrogen fixation capacity. Consequently, introduction of ‘elite’ bacteria in fields commonly does not result in crop yield increase. This review highlights that the primary regulation of N2 fixation is a result of response of integrated physiological activity at the plant level. Nitrogen feedback from the host plant plays an important role in regulating the N2 fixation rate. Rapid sequestration of fixed nitrogen by the plant is especially important for high N2 fixation activity. In addition, water cycling in the plant between the shoot and nodules plays a key role in sustaining high N2 fixation activity. Therefore, attention in selecting the host-plant genotype is suggested to be the next step to increasing N2 fixation activity of grain legumes. Grain legume, N2-fixing bacteria, nodules, soil inoculation, symbiotic nitrogen fixation, water flux Introduction The global energy crisis in the early 1970s sparked major efforts to decrease fossil fuel consumption, including in agriculture. One major target was enhanced legume N2 fixation activity so as to decrease the need for the application of nitrogen fertilizer in crop production. Hence, there were major efforts to seek solutions for increasing symbiotic N2 fixation rates (Hardy, 1994). However, no important agricultural advances resulted from this research (Henzell, 1988; Shiferaw et al., 2004). Even with nearly a half century of research, the need to decrease the dependence on nitrogen fertilizers is probably now greater than it was in the 1970s. Both the cost and the environmental impact of nitrogen fertilizer is a major concern in virtually all cropping systems. Yet, the application of the latest biological innovations, including application of molecular genetic approaches, has still not resulted in any major impact on grain legume production. In this review, it is suggested that a new perspective is needed in understanding the limits on symbiotic N2 fixation and in developing new opportunities to improve N2 fixation production by grain legumes. The underlying conclusion of this review is that the emphasis of past N2 fixation research on bacteria needs to shift to the host plant. Much of the N2 fixation research initiated nearly 50 years ago was focused on the bacteria partner in the bacteria–plant symbiosis, which at the time appeared logical and the results could seemingly be readily applied in growers’ fields. Even the recent International Congress on Nitrogen Fixation (September 2017, Granada, Spain) reflected this continuing trend with only one plenary session out of seven considering plant physiology and crop productivity. Unfortunately, the returns on bacterial research have been very modest, except for lands that were essentially virgin of commercial grain legume production. The lack of benefit from bacterial improvement has occurred because it proved to be very difficult to introduce into agricultural fields bacteria that were competitive with indigenous bacteria, able to sustain populations in natural environments, and ultimately had a capacity for up-regulation of N2 fixation rates. Some of these challenges are discussed in the first sections of this review. The role of the host plant in regulating N2 fixation activity has commonly been given minor consideration. Due to the challenges in tracking nodulation and N2 fixation activity, breeding programs have essentially disregarded plant traits that might be associated with increased N2 fixation capacity. It is becoming increasing clear, however, in more recent research on legume N2 fixation that it is the host plant that has a dominant role in regulating N2 fixation. To increase N2 fixation activity, it now appears necessary to select plant genotypes that have the growth and physiological capacity to support increased nitrogen input to the plant. The regulatory mechanisms active in plants that control N2 fixation rates and the opportunities to increase N2 fixation rates are discussed in later sections of this review. Bacteria introduction to virgin soils Soil is a very hostile and competitive environment in which microorganisms are struggling for resources in occupying specific niches. Since the natural microbial community becomes well adapted to a particular soil environment, exogenous microorganisms are seldom able to displace a native population and occupy its niche (Nazir et al., 2013). For example, a decrease of 99% in an introduced bradyrhizobial population was observed after the first cropping cycle of soybean [Glycine max (L.) Merr.] in a soil originally devoid of soybean-nodulating bradyrhizobial populations (Zilli et al., 2013). Hence, a key issue is the ability to introduce ‘elite’ bacteria that will result in the establishment of a minimum population density to alter plant–microbe interaction effectively (Barea et al., 2005). One of the challenges is the rapid adaptation of inoculated or even dispersed bacterial strains to different environments (Ferreira and Hungria, 2002; Mendes et al., 2004; Loureiro et al., 2007). The rhizobial genome is highly plastic, leading to genetic recombination that may even be accelerated under stress conditions, as in tropical soils. However, introduced bacterial strains that adapt to a new area may not necessarily be the most effective in fixing N2. For example, the strain SEMIA 566 of Bradyrhizobium japonicum has been used as an inoculant for soybean in southern Brazil since the 1960s. This strain was robust in increasing nodulation but was subsequently found in the field to be among the poorest in supporting plant nitrogen accumulation (Hungria et al., 1998). Interestingly, in a survey of soybean nodules in the northern Cerrado of Brazil, this strain was found to occupy ~70% of the nodules, although the sample site was thousands of kilometers away from the southern area where it had been used as an inoculant (Vargas et al., 1994). These observations indicated that an adapted bacterium can be highly competitive and readily dispersed by wind and rain (Ferreira and Hungria, 2002), but may contribute little or nothing to production of increased N2 fixation. Not surprisingly, therefore, there are large differences between the N2 fixation potential of microorganisms selected under controlled conditions and their performance in real-world conditions. There are several examples of failures in the performance of elite bacteria when introduced in the field (Dobbelaere et al., 2001). Studies on the selection of rhizobial strains for tolerance to high temperatures have not correlated with field responses (Hungria and Vargas, 2000). The lack of effect can be explained by a myriad of interactions at the chemical (e.g. pH, availability of nutrients, energy sources, and salinity), physical (e.g. availability of water, temperature, and aeration), and biological (e.g. antibiosis, predation, parasitism, etc.) levels. Several adverse factors may lead to a progressive decline of introduced microorganisms, thus impairing the promising responses observed under controlled conditions (O’Callaghan et al., 2001; Strigul and Kravchenko, 2006). Nevertheless, there is some encouragement about the possibility of bacterial inoculation into soils initially devoid of soybean-nodulating rhizobia over successive soybean (Glycine max) cropping cycles (Galli-Terasawa et al., 2003; Silva-Batista et al., 2007). In the study by Mendes et al. (2004), with inoculation of a soil without indigenous bradyrhizobia in four successive years, the number of soybean-nodulating rhizobia was 103 bacteria g–1 soil after three cropping cycles, increasing to 105 bacteria g–1 soil after five cropping cycles in a soil initially devoid of soybean bradyrhizobia. Introduction of bacteria to agricultural soils Bacterial inoculation of soils on which soybean has been previously grown has generally resulted in little or no increase in nodulation or crop yield (Elkins et al., 1976; Schulz and Thelen, 2008; De Bruin et al., 2010; Mason et al., 2016). Specifically, attempts to introduce elite rhizobia populations into soils with established bradyrhizobia populations have shown little success. A mutant bradyrhizobia bacteria line with 100% greater specific N2 fixation activity than the wild type in controlled environments showed no soybean yield increase as compared with indigenous bacteria in a 2 year field test (Williams and Phillips, 1983). In field tests of bradyrhizobium strains with the Hup+ characteristic for improved energy efficiency in the nodule, there was no yield increase as compared with strains without the characteristic (Hup–) (Hume and Shelp, 1990). There is little convincing evidence that introduction into the field of genetically manipulated bacteria has resulted in yield increase (Thies et al., 1991; Keyser and Li, 1992). Again, genetic stability of introduced bacterial strains is a problem when inoculations are applied to agricultural soils. In Brazil, tests showed substantial bacterial mutation when bacteria were re-isolated from Brazilian soils several years after inoculation. In the southern region of Brazil, 38% of the isolates obtained after 17 years were unknown in relation to the original inoculated strain (Ferreira et al., 2000), whereas in the tropical region of the Cerrados, the number of isolated strains unrelated to the originally inoculated strains was 50% in the same period (Galli-Terasawa et al., 2003; Silva-Batista et al., 2007). Annual re-inoculation of bacteria in Brazil at sowing with more effective N2-fixing strains is necessary to compete with the established strains and increase the nodule occupancy by more effective strains. In fact, the inoculation of a highly saprophytic and effective N2 fixation strain (SEMIA 5079=CPAC 15) was a strategy to prevent the colonization by competitive but less effective N2-fixing bacteria strains in new soybean areas, especially in the Cerrados (Mendes et al., 2004). Overall, inoculation of soils containing established populations of rhizobia has proven to show little or no response. In the citations above, experimental results showed that annual inoculation (or re-inoculation) of soybean in Brazil and Argentina has resulted in an average gain in yields of only 6–8%, and responses in the USA have been mostly neutral. The small but significant benefit gained in Brazil might occur to overcome decreasing populations of introduced bacteria in the soil due to the harsh environmental conditions, particularly water deficit, during the period between growing seasons. For this reason, annual inoculation with elite bacterial strains is suggested in Brazil even thought very modest yield increases are expected. Host regulation of N2 fixation rate In addition to the challenge of establishing new bacteria in the soil, there is increasing evidence that the role of bacteria in determining the N2 fixation rate may be overshadowed by processes dependent on the host plant. There are several possibilities to explain the dominant role of the host plant in regulating the N2 fixation rate. Briefly reviewed here are regulatory hypotheses related to the flow of oxygen, nitrogenous compounds, and water in the nodule and plant. Bacteria-derived bacteriods, which provide nitrogenase to catalyze the primary step in gaseous nitrogen fixation, are located in special cells in the interior volume of nodules (Fig. 1). Since nitrogenase is inactivated by oxygen, nodules provide a special structure within which oxygen is maintained at suitably low concentrations. Much of the regulation of oxygen concentration occurs at the inner cortex of the nodules in a continuous layer of two or three cells immediately surrounding the central volume (Minchin, 1997). The oxygen permeability of the inner cortex varies in response to various conditions (e.g. Weisz et al., 1985) so that the oxygen concentration in the interior nodule is fairly stable in the range of 5–60 μmol m–3 (Millar et al., 1995). Consequently, the adjustments in the permeability of the oxygen barrier are one possibility for regulation by the host plant of the N2 fixation rate (Serraj et al., 1999; Sulieman and Tran, 2013). Fig. 1. View largeDownload slide Cross-section of a soybean nodule and root. The inner cortex constituted of a few cell layers is the site of oxygen root regulation into the interior of the nodules. Fig. 1. View largeDownload slide Cross-section of a soybean nodule and root. The inner cortex constituted of a few cell layers is the site of oxygen root regulation into the interior of the nodules. It is not clear, however, whether adjustment in the oxygen permeability of the inner cortex is the primary regulator of N2 fixation or whether the permeability adjustments are in response to triggers from other variables in the nodule. Two frequently considered prime regulators are the availability of carbon resources or accumulation of nitrogen products in the nodule. However, most evidence now indicates that carbon availability in nodules is commonly not limiting the N2 fixation rate (Gonzalez et al., 2015). Much more emphasis to explain regulation of nodule N2 fixation has ben given to the accumulation of nitrogenous compounds in nodules as invoking potential feedback control of the N2 fixation rate. The accumulation of nitrogenous compounds can result either from synthesis in the nodules or from transport from the plant shoot (Fig. 2). Initial experiments involving the feeding of plants with inorganic nitrogen supported the idea of transported nitrogenous compounds being of potential importance. Neo and Layzell (1997) reported decreased N2 fixation activity that was associated with increased amino acids and amides in the phloem sap. Similar decreased N2 fixation activity was found with accumulation of amino acids and amides in nodules (Bacanamwo and Harper, 1997; Sulieman et al., 2010). Fig. 2. View largeDownload slide Schematic of flows between nodules and shoots (Serraj et al., 2001). ALN, allantoin; ALAC, allantoic acid; Asn, asparagin; Nase, nitrogenase. Fig. 2. View largeDownload slide Schematic of flows between nodules and shoots (Serraj et al., 2001). ALN, allantoin; ALAC, allantoic acid; Asn, asparagin; Nase, nitrogenase. Vadez et al. (2000) reported feeding experiments with soybean plants in which hydroponic solutions were supplied with increased concentrations of asparagine or allantoic acids. In these feeding experiments, N2 fixation activity was depressed and nodule concentrations of asparagine, aspartate, and uriedes were increased. King and Purcell (2005) subjected soybean plants to a water deficit treatment that resulted in substantial decreases in the N2 fixation rate and in increases in concentrations of nodule asparagine, aspartate, and uriedes. However, 2 d after re-watering plants, King and Purcell (2005) observed that N2 fixation rates had nearly fully recovered, while nodule aspartate and especially asparagine levels were still at high levels. They concluded that asparagine was not the primary inhibitory signal for N2 fixation. In contrast, however, Sulieman and Tran (2013) concluded in a review on regulation of N2 fixation that asparagine was a likely candidate for affecting the nitrogen feedback response. There is as of yet no general consensus about the specific nitrogen compound that could be the critical feedback regulator of N2 fixation rate. An option for regulation of nodule N2 fixation, which has not been considered in detail previously, is based on water flux to and from nodules. Nitrogen-fixing nodules are somewhat unusual in that nearly all the water that flows into the nodule is a result of plant phloem flow (Walsh, 1995). Therefore, the amount of water flowing from nodules in the xylem is intimately linked to phloem flow into the nodules. Any plant disruption resulting in decreased phloem flow, and consequently decreased xylem flow from the nodule, may result in a cascade of changes in nodules. These changes could include availability of carbon in the nodule, accumulation in the nodules of various nitrogenous compounds, and decreased turgor of the inner cortex cells resulting in decreased oxygen diffusion into the nodule. An additional sensitivity to decreased water flow to the nodule could be a loss of turgor in the vascular parachyma cells that are responsible for transporting amino acids and/or ureides into the apoplast for loading of the nodule xylem (Carter and Tegeder, 2016). Loss of turgor could account directly for less loading of the xylem (Wolswinkel and Ammerlaan, 1984) and an accumulation of nitrogenous compounds in the nodule. Dependence of nodule activity on phloem flow offers a potential explanation of the high sensitivity of N2 fixation activity to soil drying often observed in grain legumes (Sinclair and Vadez, 2012; Gonzalez et al., 2015). Phloem flow is highly dynamic and depends on several factors. One important factor can be the exchange of water within the vascular bundles in the stem and root between the phloem and xylem. The two vascular elements are not hydrologically isolated from each other so that water moves from the higher hydrostatic pressure of the phloem to the lower hydrostatic pressure of the xylem (Sevanto, 2014). The amount of water flow depends on hydrological conductance between cells and on the hydrostatic pressure gradient between the two vascular tissues. As the xylem hydrostatic pressure in the roots and stem decreases with soil drying, the increased gradient between the phloem and xylem in the root and stem will result in increased water flow to the xylem from the phloem. The loss of water from the phloem will hypothetically result in decreased phloem flow into the nodule, and, consequently, trigger changes in the nodule resulting in decreased N2 fixation activity. The above hypothesis also allows the possibility that N2 fixation activity is more sensitive to soil drying than the transpiration rate. Nitrogen fixation as proposed in the above hypothesis is sensitive to gradients in hydrostatic potential between adjacent phloem and xylem cells in the stem and root, while transpiration rate sensitivity to soil drying is dependent on the overall hydraulic potential gradient from the soil to guard cells. The transpiration rate decreases when the hydraulic conductance in the water pathway, especially the decreasing hydraulic conductance of the soil (Sinclair, 2005), decreases to a threshold where water flow in the soil and through the plant cannot match the ambient transpiration rate. Hence, the N2 fixation rate and transpiration rate are dependent on different hydraulic gradients and conductances: N2 fixation is dependent on the hydraulic gradient and conductance between adjacent phloem and xylem cells in the stem and root, and transpiration is dependent on the overall hydraulic gradient and conductance in the pathway from the soil to guard cells. If decreasing xylem hydrostatic pressure causes increased water transfer from the phloem to the xylem with soil drying before the threshold soil water content causing the decreased transpiration rate, then the conditions are in place for expression of differential sensitivity to soil drying. Unfortunately, there is at this time no direct experimental evidence to resolve this hypothesis, although Purcell and Sinclair (1995) found that addition of polyethylene glycol to the root solution below the nodulation zone resulted in decreased acetylene reduction activity within 4 h after treatment. One attribute of the phloem flow-based hypothesis influencing N2 fixation activity is that it is consistent with ‘local’ control of the N2 fixation rate observed in split-root experiments (Gil-Quintana et al., 2013). The N2 fixation activity of nodules attached to roots in the well-watered split of the split-root system was observed to be unaffected by soil drying of the water-deficit split. However, N2 fixation of the water-deficit split was decreased, which could be predicted as a result of increased water flow from the phloem to the xylem in the roots in the water-deficit split. The water loss from the phloem to the xylem in the water-deficit roots would probably result in decreased water flow to the nodules of the water-deficit split and, as discussed above, this could lead to the cascade of changes in the nodule resulting in decreased N2 activity. ‘Local’ control from the perspective of the phloem flow hypothesis can be interpreted as a response to differences in phloem flow to specific nodules. Increasing N2 fixation input Non-stressed conditions Given that low nitrogen concentrations in the phloem feeding back to nodules seems critical to sustaining high N2 fixation rates, increasing N2 fixation rates may be dependent on mechanisms that sequester nitrogen in the plant shoot and minimize nitrogen feedback to nodules. That is, a high ‘demand’ for nitrogen by the host plant is necessary to avoid a nitrogen feedback signal to the nodules. Nitrogen demand can be maintained at a high level either by establishing a high concentration of sequestered nitrogen in the plant shoot components, or by increasing the total size of the plant. Increasing plant size may be an especially rewarding approach to increase N2 fixation activity, and may contribute in other ways in plant selection for increased yield. A high correlation has been found between plant leaf area and N2 fixation activity, and between shoot mass and N2 fixation activity measured on soybean plants growing in the field (Denison et al., 1985). Early plant vigor with rapid leaf area development is a direct approach to increase plant size and nitrogen demand. An extended vegetative phase also allows for a period of greater nitrogen accumulation in the plant. Increased plant density could also result in a greater plant mass per unit land area, and hence, greater potential for N2 fixation activity per unit land area. In the experiment of De Luca et al. (2014) decreasing soybean plant density in the field from 32 plants m–2 to 8 plants m–2 resulted in similar grain yield so that N2 fixation activity per plant was able to increase at least 4-fold at the lower density as compared with the high density. In farming systems where a cereal crop following a grain legume crop may depend to a large extent on residual organic nitrogen from the legume crop, legume N2 fixation activity might be enhanced by sustaining high N2 fixation rates during seed growth. Commonly, once seeds start to grow rapidly on grain legume plants, there is a decrease in N2 fixation activity, presumably due to an eventual decrease in photosynthate transport to nodules. If plant alterations were made to limit seed growth so more photosynthate is delivered to nodules, then overall N2 fixation activity might be sustained at a higher rate into the reproductive growth period (Denison and Sinclair, 1985) and increase the overall amount of fixed nitrogen. Of course, this approach to increasing N2 fixation would probably result in less photosynthate for seeds and, hence, lower grain legume seed yield. However, the loss in seed yield might be offset by the greater amounts of nitrogen accumulation in the vegetative plant resulting in vegetative mass with greater nitrogen concentration for higher quality fodder for animals or green manure for the following cereal crop (Sinclair and Vadez, 2012). An additional strategy to increase crop N2 fixation input, co-inoculation with Azospirillum, has proven to be promising in stimulating N2 fixation activity in legume crops (Hungria et al., 2013; Souza and Ferreira, 2017). Plant growth-promoting Azospirillum bacteria when sprayed on rhizobia-inoculated common bean (Phaseolus vulgaris L.) as compared with only rhizobia inoculation resulted in a 31% increase in shoot mass (Souza and Ferreira, 2017), which may have stimulated up-regulation of the N2 fixation rate. Nodule mass was increased by 25% and seed yield was increased by 26%. In soybean, it was found in the field that the combination of Azospirillum and Bradyrhizobia as compared to solely bradyrhizobia treatment resulted in increased nitrogen accumulation but shoot mass accumulation was unaffected, and there was no response under sterile greenhouse conditions (Chibeba et al., 2015). Water-deficit conditions As discussed previously, accumulation of the products of N2 fixation in nodules can invoke a depression in the N2 fixation rate. As hypothesized earlier, decreased phloem flow from the host plant to the nodules can result in a decreased delivery of water that in turn may limit removal of nitrogen products from the nodules. In soybean and cowpea (Vigna unguiculata), where N2 fixation rates are commonly high, rapid transport of nitrogenous products from the nodules may be especially important. Therefore, high amounts of water cycling through the nodules are likely to be needed to avoid limitation on N2 fixation activity. This is consistent with the high sensitivity of N2 fixation in these species to soil drying (Sinclair and Serraj, 1995; Sinclair et al., 2015). Figure 3b illustrates that the N2 fixation rate commonly decreases in soybean at high soil water content, defined in this case as the fraction of transpirable soil water remaining in the soil. Fig. 3. View largeDownload slide Response of N2 fixation activity to the fraction transpirable soil water (FTSW) found (a) in N2 fixation drought-tolerant soybean genotype PI 461938, and (b) in cultivar Benning, which is representative of commonly observed non-tolerant N2 fixation (Devi and Sinclair, 2013). Fig. 3. View largeDownload slide Response of N2 fixation activity to the fraction transpirable soil water (FTSW) found (a) in N2 fixation drought-tolerant soybean genotype PI 461938, and (b) in cultivar Benning, which is representative of commonly observed non-tolerant N2 fixation (Devi and Sinclair, 2013). A major effort has been underway in the USA to minimize the impact of drought on soybean N2 fixation productivity. Early on it was discovered that the cultivar ‘Jackson’ exhibited more drought tolerance in its N2 fixation activity than all other tested cultivars (Sall and Sinclair, 1991), which was associated with lower ureide concentration in the shoots than other genotypes (King and Purcell, 2005; Charlson et al., 2009). A breeding program using Jackson as a parent led to the release of two high yield, high N2 fixing, drought-tolerant lines (Chen et al., 2007). More recently, genotype ‘PI 471938’ was discovered to exhibit even greater N2 fixation drought tolerance than Jackson (Fig. 3a) (Devi and Sinclair, 2013). Several elite breeding lines with N2 fixation drought tolerance have now been developed using PI 471938 as a parent (Devi et al., 2014), and one line has been recently released as a variety (Carter et al., 2016). The techniques developed in these past studies offer approaches for further development of N2 fixation drought tolerance in soybean, and in other legume species (Sinclair et al., 2015). Simulation studies have indicated that increasing N2 fixation drought tolerance in soybean will probably result in yield increases across the USA and Africa. The probability of yield increase was ≥85% in the 50 simulated years at nearly all locations in the USA (Fig. 4) (Sinclair et al., 2010). In Africa, yield increase was simulated for ≥85% of the years in many locations and for ≥70% for most of the locations (Sinclair et al., 2014). Fig. 4. View largeDownload slide Simulated soybean yield response to increasing N2 fixation tolerance to soil drying (Sinclair et al., 2010). (a) The probability of yield increase over the 50 years of simulations. The absolute yield increase simulated for each gird location at (b) 75%, (c) median, and (d) 25% percentile ranking of yield change at each grid location. Fig. 4. View largeDownload slide Simulated soybean yield response to increasing N2 fixation tolerance to soil drying (Sinclair et al., 2010). (a) The probability of yield increase over the 50 years of simulations. The absolute yield increase simulated for each gird location at (b) 75%, (c) median, and (d) 25% percentile ranking of yield change at each grid location. Phosphorus-deficient conditions Phosphorus deficiency severely retards both plant growth and N2 fixation activity in grain legumes. There are two apparent methods whereby phosphorus deficiency would result in low N2 fixation activity. Since nodules are sites of high rates of energy transfer to support N2 fixation, low levels of phosphorus in the nodules could seemingly result directly in low N2 fixation rates. The second method of limitation could be a feedback mechanism in which low shoot growth as a result of low plant phosphorus intake results in a decreased demand for nitrogen. Nodules, in fact, under sufficient phosphorus conditions commonly have the highest phosphorus concentrations in the plant (Pereira and Bliss, 1987). Also, nodules are able to maintain high levels of phosphorus under phosphorus-deficient conditions except for extreme deficiency treatments (Pereira and Bliss, 1987; Sa and Israel, 1991; Kouas et al., 2005; Miao et al., 2007; Sulieman and Tran, 2015). In addition, even under severe phosphorus deficiency, Sa and Israel (1991) did not find a decrease in the energy charge or ATP concentration in the bacteroid fraction of soybean nodules, and only comparatively small decreases for whole nodules. Not surprisingly, the potential specific N2 fixation rate was little affected by phosphorus deficiency (Sa and Israel, 1991; Vadez et al., 1997). Therefore, there appears to be little evidence that a phosphorus deficiency has a direct metabolic role in nodules in limiting N2 fixation capacity. It appears that the main influence of phosphorus deficiency on the N2 fixation rate may be a feedback regulation through elevated nitrogen levels as a result of low plant growth. Under low phosphorus conditions, plant growth is severely limited even though nodule phosphorus levels are decreased little or not at all (Pereira and Bliss, 1987; Kouas et al., 2005). In a comparison of common bean genotypes, Vadez et al. (1999) found under phosphorus deficiency that genotypic variability in the amount of nitrogen fixed was closely correlated with the mass of various plant components. Ribet and Drevon (1995) found in soybean that N2 fixation capacity per unit nodule mass was unchanged under severe phosphorus deficiency even though both shoot and nodule mass were greatly decreased. Bargaz et al. (2011) observed that under phosphorus-deficient conditions, nodule oxygen permeability was decreased, indicating the possibility of feedback limitation on the N2 fixation rate. To overcome the phosphorus limitation if possible, selection of plants with enhanced overall plant growth under phosphorus deficiency seems to be a viable approach to minimize a nitrogen feedback limitation in the nodules. Conclusions There appears to be a major role in the regulation of N2 fixation activity for integrated processes expressed in the host plant. The whole-plant system can influence the N2 fixation rate as a result of water and nitrogen flow to the nodules in the phloem, and nitrogenous compounds flowing from nodules in the xylem. The nitrogen feedback to nodules can seemingly be minimized by increasing the overall plant demand for nitrogen that results in nitrogen sequestration in the plant shoot. Since nitrogen feedback in nodules is hypothesized to be a critical regulation point, the phloem flow rate to deliver water to nodules for supporting water flow from nodule in the xylem could be an important control point. Disruptions of phloem flow to nodules can result in a cascade of changes in nodules that may include accumulation of nitrogenous compounds in the nodules associated with a decreased N2 fixation rate. The great sensitivity to soil drying exhibited by some grain legumes may also be a result of decreased phloem flow to nodules and accumulation of nitrogenous compounds. A better understanding of the dynamics of phloem flow and the water budget in the plant could lead to new approaches to increase N2 fixation rates of grain legumes under both under non-stressed and drought conditions. References Bacanamwo M , Harper JE . 1997 . The feedback mechanism of nitrate inhibition of nitrogenase activity in soybean may involve asparagine and/or products of its metabolism . Physiologia Plantarum 100 , 371 – 377 . Google Scholar CrossRef Search ADS Bargaz A , Drevon J-J , Oufdou K , Btissam-Mandri , Faghire M , Ghoulam C . 2011 . Nodule phosphorus requirement and O2 uptake in common bean genotypes under phosphorus deficiency . Soil and Plant Science 61 , 602 – 611 . Barea JM , Pozo MJ , Azcón R , Azcón-Aguilar C . 2005 . Microbial co-operation in the rhizosphere . Journal of Experimental Botany 56 , 1761 – 1778 . Google Scholar CrossRef Search ADS PubMed Carter AM , Tegeder M . 2016 . Increasing nitrogen fixation and seed development in soybean requires complex adjustments of nodule nitrogen metabolism and partitioning processes . Current Biology 26 , 2044 – 2051 . Google Scholar CrossRef Search ADS PubMed Carter TE Jr , Todd SM , Gillen AM . 2016 . Registration of ‘USDA-N8002’ soybean cultivar with high yield and abiotic stress resistance traits . Journal of Plant Registrations 10 , 238 – 245 . Google Scholar CrossRef Search ADS Charlson DV , Korth KL , Purcell LC . 2009 . Allantoate amidohydrolase transcript expression is independent of drought tolerance in soybean . Journal of Experimental Botany 60 , 847 – 851 . Google Scholar CrossRef Search ADS PubMed Chen P , Sneller CH , Purcell LC , Sinclair TR , King CA , Ishibashi T . 2007 . Registration of soybean germplasm lines R01-416F and R01-581F for improved yield and nitrogen fixation under drought stress . Journal of Plant Registrations 1 , 166 – 167 . Google Scholar CrossRef Search ADS Chibeba AM , Guimarães MF , Brito OR , Nogueira MA , Araujo RS , Hungria M . 2015 . Co-inoculation of soybean with Bradyrhizobium and Azospirillum promotes early nodulation . American Journal of Plant Sciences 6 , 1641 – 1649 . Google Scholar CrossRef Search ADS De Bruin JL , Pedersen P , Conley SP , Gaska JM , Naeve SL , Kurle JE , Elmore RW , Giesler LJ , Abendroth LJ . 2010 . Probabilitiy of yield response to inoculants in fields with a history of soybean . Crop Science 50 , 263 – 272 . de Luca MJ , Nogueira MA , Hungria M . 2014 . Feasibility of lowering soybean planting density without compromising nitrogen fixation and yield . Agronomy Journal 106 , 2118 – 2124 . Google Scholar CrossRef Search ADS Denison RF , Sinclair TR . 1985 . Diurnal and seasonal variation in dinitrogen fixation (acetylene reduction) rates by field-grown soybean . Agronomy Journal 77 , 679 – 684 . Google Scholar CrossRef Search ADS Denison RF , Weisz PR , Sinclair TR . 1985 . Variability among plants in dinitrogen fixation (acetylene reduction) rates by field-grown soybean . Agronomy Journal 77 , 947 – 950 . Google Scholar CrossRef Search ADS Devi MJ , Sinclair TR . 2013 . Nitrogen fixation drought tolerance of the slow wilting soybean PI 471938 . Crop Science 53 , 2072 – 2078 . Google Scholar CrossRef Search ADS Devi MJ , Sinclair TR , Chen P , Carter TE . 2014 . Evaluation of elite southern maturity soybean breeding lines for drought-tolerant traits . Agronomy Journal 106 , 1947 – 1954 . Google Scholar CrossRef Search ADS Dobbelaere S , Croonenborghs A , Thys A , et al. 2001 . Responses of agronomically important crops to inoculation with Azospirillum . Australian Journal of Plant Physiology 28 , 871 – 879 . Elkins DM , Hamilton G , Chan CKY , Briskovich MA , Vandeventer JW . 1976 . Effect of cropping history on soybean growth and nodulation and soil rhizobia . Agronomy Journal 68 , 513 – 517 . Google Scholar CrossRef Search ADS Ferreira MC , Andrade DS , Chueire LMO , Takemura SM , Hungria M . 2000 . Tillage method and crop rotation effects on the population sizes and diversity of bradyrhizobia nodulating soybean . Soil Biology and Biochemistry 32 , 627 – 637 . Google Scholar CrossRef Search ADS Ferreira MC , Hungria M . 2002 . Recovery of soybean inoculant strains from uncropped soils in Brazil . Field Crops Research 79 , 139 – 152 . Google Scholar CrossRef Search ADS Galli-Terasawa LV , Glienke-Blanco C , Hungria M . 2003 . Diversity of soybean rhizobial population adapted to a Cerrados soil . World Journal of Microbiology and Biotechnology 19 , 933 – 939 . Google Scholar CrossRef Search ADS Gil-Quintana E , Larrainzar E , Seminario A , Díaz-Leal JL , Alamillo JM , Pineda M , Arrese-Igor C , Wienkoop S , González EM . 2013 . Local inhibition of nitrogen fixation and nodule metabolism in drought-stressed soybean . Journal of Experimental Botany 64 , 2171 – 2182 . Google Scholar CrossRef Search ADS PubMed Gonzalez EM , Larrainzar E , Marino D , Wienkoop S , Gil-Quintana E , Arrese-Igor C . 2015 . Physiological responses of N2-fixing legumes to water limitation . In: Sulieman S , Tran L-SP , eds. Legume nitrogen fixation in a changing environment . Switzerland : Springer , 5 – 33 . Google Scholar CrossRef Search ADS Hardy RWF . 1994 . Biological nitrogen fixation: research challenges . Washington, DC : National Academy Press . Henzell EF . 1988 . The role of biological nitrogen fixation research in solving problems in tropical agriculture . Plant and Soil 108 , 15 – 21 . Google Scholar CrossRef Search ADS Hume DJ , Shelp BJ . 1990 . Superior performance of the HUP– Bradyrhizobium japonicum strain 532C in Ontario soybean field trials . Canadian Journal of Plant Science 70 , 661 – 666 . Google Scholar CrossRef Search ADS Hungria M , Boddey LH , Santos MA , Vargas MAT . 1998 . Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elkanii strains . Biology and Fertility of Soils 27 , 393 – 399 . Google Scholar CrossRef Search ADS Hungria M , Campo RJ , Mendes IC , Graham PH . 2006 . Contribution of biological nitrogen fixation to the nitrogen nutrition of grain crops in the tropics: the success of soybean (Glycine max L. Merr.) in South America . In: Singh RP , Shankar N , Jaiwal PK , eds. Nitrogen nutrition in plant productivity . Houston, TX : Studium Press , 43 – 93 . Hungria M , Nogueira MA , Araujo RS . 2013 . Co-inoculation of soybeans and common beans with rhizobia and azospirilla: strategies to improve sustainability . Biology and Fertility of Soils 49 , 791 – 801 . Google Scholar CrossRef Search ADS Hungria M , Vargas MAT . 2000 . Environmental factors affecting N2 fixation in grain legumes in the tropics, with an emphasis on Brazil . Field Crops Research 65 , 151 – 164 . Google Scholar CrossRef Search ADS Keyser HH , Li F . 1992 . Potential for increasing biological nitrogen fixation in soybean . Plant and Soil 141 , 119 – 135 . Google Scholar CrossRef Search ADS King CA , Purcell LC . 2005 . Inhibition of N2 fixation in soybean is associated with elevated ureides and amino acids . Plant Physiology 137 , 1389 – 1396 . Google Scholar CrossRef Search ADS PubMed Kouas S , Labidi N , Debez A , Abdelly C . 2005 . Effect of P on nodule formation and N fixation in bean . Agronomy for Sustainable Development 25 , 389 – 393 . Google Scholar CrossRef Search ADS Loureiro MF , Kaschuk G , Alberton O , Hungria M . 2007 . Soybean [Glycine max (L.) Merrill] rhizobial diversity in Brazilian oxisols under various soil, cropping, and inoculation managements . Biology and Fertility of Soils 43 , 665 – 674 . Google Scholar CrossRef Search ADS Mason S , Galusha T , Kmail Z . 2016 . Soybean yield and nodulation response to crop history and inoculation . Agronomy Journal 108 , 309 – 312 . Google Scholar CrossRef Search ADS Mendes IC , Hungria M , Vargas MAT . 2004 . Establishment of Bradyrhizobium japonicum and B. elkanii strains in a Brazilian Cerrado oxisol . Biology and Fertility of Soils 40 , 28 – 35 . Google Scholar CrossRef Search ADS Miao S-J , Qiao Y-F , Han X-Z , An M . 2007 . Nodule formation and development in soybeans (Glycine max L.) in response to phosphorus supply in solution culture . Pedosphere 17 , 36 – 43 . Google Scholar CrossRef Search ADS Millar AH , Day DA , Bergersen FJ . 1995 . Microaerobic respiration and oxidative phosphorylation by soybean nodule mitochondria: implications for nitrogen fixation . Plant, Cell and Environment 18 , 715 – 726 . Google Scholar CrossRef Search ADS Minchin FR . 1997 . Regulation of oxygen diffusion in legume nodules . Soil Biology and Biochemistry 29 , 881 – 888 . Google Scholar CrossRef Search ADS Nazir R , Semenov AV , Sarigul N , van Elsas JD . 2013 . Bacterial community establishment in native and non-native soils and the effect of fungal colonization . Microbiology Discovery 1 , 8 . Google Scholar CrossRef Search ADS Neo HH , Layzell DB . 1997 . Phloem glutamine and the regulation of O2 diffusion in legume nodules . Plant Physiology 113 , 259 – 267 . Google Scholar CrossRef Search ADS PubMed O’Callaghan M , Gerard EM , Johnson VW . 2001 . Effect of soil moisture and temperature on survival of microbial control agents . New Zealand Plant Protection 54 , 128 – 135 . Pereira PAA , Bliss FA . 1987 . Nitrogen fixation and plant growth of common bean (Phaseolus vulgaris L.) at different levels of phosphorus availability . Plant and Soil 104 , 79 – 84 . Google Scholar CrossRef Search ADS Purcell LC , Sinclair TR . 1995 . Nodule gas exchange and water potential response to rapid imposition of water deficit . Plant, Cell and Environment 18 , 179 – 187 . Google Scholar CrossRef Search ADS Ribet J , Drevon J-J . 1995 . Increase in permeability to oxygen and oxygen uptake in soybean nodules under limiting phosphorus nutrition . Physiologia Plantarum 94 , 298 – 304 . Google Scholar CrossRef Search ADS Sa TM , Israel DW . 1991 . Energy status and functioning of phosphorus-deficient soybean nodules . Plant Physiology 97 , 928 – 935 . Google Scholar CrossRef Search ADS PubMed Sall K , Sinclair TR . 1991 . Soybean genotypic differences in sensitivity of symbiotic nitrogen fixation to soil dehydration . Plant and Soil 133 , 31 – 37 . Google Scholar CrossRef Search ADS Schulz TJ , Thelen KD . 2008 . Soybean seed inoculants and fungicidal seed treatment effects on soybean . Crop Science 48 , 1975 – 1983 . Google Scholar CrossRef Search ADS Serraj R , Sinclair TR , Purcell LC . 1999 . Symbiotic N2 fixation response to drought . Journal of Experimental Botany 50 , 143 – 155 . Serraj R , Vadez V , Sinclair TR . 2001 . Feedback regulation of symbiotic N2 fixation under drought stress . Agronomie 21 , 621 – 626 . Google Scholar CrossRef Search ADS Sevanto S . 2014 . Phloem transport and drought . Journal of Experimental Botany 65 , 1751 – 1759 . Google Scholar CrossRef Search ADS PubMed Shiferaw B , Bantilian MCS , Serraj R . 2004 . Harnessing the potential of BNF for poor farmers: technological, policy and institutional constraints and research needs . In: Serraj R , ed. Symbiotic nitrogen fixation: prospects for enhanced application in agriculture . New Delhi : Science Publishers , 3 . Silva-Batista JS , Hungria M , Barcellos FG , Ferreira MC , Mendes IC . 2007 . Variability in Bradyrhizobium japonicum and B. elkanii seven years after introduction of both the exotic microsymbiont and the soybean host in a cerrados soil . Microbial Ecology 53 , 270 – 284 . Google Scholar CrossRef Search ADS PubMed Sinclair TR . 2005 . Theoretical analysis of soil and plant traits influencing daily plant water flux on drying soils . Agronomy Journal 97 , 1148 – 1152 . Google Scholar CrossRef Search ADS Sinclair TR , Manandhar A , Belko N , Riar M , Vadez V , Roberts PA . 2015 . Variation among cowpea genotypes in sensitivity of transpiration rate and symbiotic nitrogen fixation to soil drying . Crop Science 55 , 2270 – 2275 . Google Scholar CrossRef Search ADS Sinclair TR , Marrou H , Soltani A , Vadez V . 2014 . Soybean production potential in Africa . Global Food Security 3 , 31 – 40 . Google Scholar CrossRef Search ADS Sinclair TR , Messina CD , Beatty A , Samples M . 2010 . Assessment across the United States of the benefits of altered soybean drought traits . Agronomy Journal 102 , 475 – 482 . Google Scholar CrossRef Search ADS Sinclair TR , Serraj R . 1995 . Legume nitrogen fixation and drought . Nature 378 , 344 . Google Scholar CrossRef Search ADS Sinclair TR , Vadez V . 2012 . The future of grain legumes in cropping systems . Crop and Pasture Science 63 , 501 – 512 . Google Scholar CrossRef Search ADS Souza JEB , Ferreira EPB . 2017 . Improving sustainability of common bean production systems by co-inoculating rhizobia and azospirilla . Agriculture, Ecosystems and Environment 237 , 250 – 257 . Google Scholar CrossRef Search ADS Strigul N , Kravchenko V . 2006 . Mathematical modeling of PGPR inoculation into the rhizosphere . Environmental Modelling and Software 21 , 1158 – 1171 . Google Scholar CrossRef Search ADS Sulieman S , Fischinger SA , Gresshoff PM , Schulze J . 2010 . Asparagine as a major factor in the N-feedback regulation of N2 fixation in Medicago truncatula . Physiologia Plantarum 140 , 21 – 31 . Google Scholar CrossRef Search ADS PubMed Sulieman S , Tran LS . 2013 . Asparagine: an amide of particular distinction in the regulation of symbiotic nitrogen fixation of legumes . Critical Reviews in Biotechnology 33 , 309 – 327 . Google Scholar CrossRef Search ADS PubMed Sulieman S , Tran LS . 2015 . Phosphorus homeostasis in legume nodules as an adaptive strategy to phosphorus deficiency . Plant Science 239 , 36 – 43 . Google Scholar CrossRef Search ADS PubMed Thies JE , Singleton PW , Bohlool BB . 1991 . Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes . Applied and Environmental Microbiology 57 , 19 – 28 . Google Scholar PubMed Vadez V , Lasso JH , Beck DP , Drevon J-J . 1999 . Variability of N2-fxaition in common bean (Phaseolus vulgaris L.) under P deficiency is related to P use efficiency . Euphytica 106 , 231 – 242 . Google Scholar CrossRef Search ADS Vadez V , Beck DP , Lasso JH , Drevon J-J . 1997 . Utilization of the acetylene reduction assay to screen for tolerance of symbiotic N2 fixation to limiting P nutrition in common bean . Physiologia Plantarum 99 , 227 – 232 . Google Scholar CrossRef Search ADS Vadez V , Sinclair TR , Serraj R . 2000 . Asparagine and ureide accumulation in nodules and shoots as feedback inhibitors of N2 fixation in soybean . Physiologia Plantarum 110 , 215 – 223 . Google Scholar CrossRef Search ADS Vargas MAT , Mendes IC , Suhet AR , Peres JRR . 1994 . Inoculation of soybean in Cerrado soils with established populations of Bradyrhizobium japonicum . Revista de Microbiologia 25 , 245 – 250 . Walsh KB . 1995 . Physiology of the legume nodule and its response to stress . Soil Biology and Biochemistry 27 , 637 – 655 . Google Scholar CrossRef Search ADS Weisz PR , Denison RF , Sinclair TR . 1985 . Response to drought stress of nitrogen fixation (acetylene reduction) rates by field-grown soybeans . Plant Physiology 78 , 525 – 530 . Google Scholar CrossRef Search ADS PubMed Williams LE , Phillips DA . 1983 . Increased soybean producitivity with a Rhizobium japonicum mutant . Crop Science 23 , 246 – 250 . Google Scholar CrossRef Search ADS Wolswinkel P , Ammerlaan A . 1984 . Turgor-sensitive sucrose and amino acid transport into developing seeds of Pisum sativum. Effect of a high sucrose or mannitol concentraion in experiments with empty ovules . Physiologia Plantarum 61 , 172 – 182 . Google Scholar CrossRef Search ADS Zilli JE , Pereira GMD , França-Júnior I , Silva K , Hungria M , Rouws JRC . 2013 . Dinâmica de rizóbios em solo do cerrado de Roraima durante o período de estiagem . Acta Amazonica 43 , 153 – 160 . Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: firstname.lastname@example.org This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
Journal of Experimental Botany – Oxford University Press
Published: Mar 24, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera