Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms

Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis. Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e. carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products. The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean. Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype. It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits. The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity. The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity. Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis. The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.     

The soybean GmN6L gene encodes a late nodulin expressed in the infected zone of nitrogen-fixing nodules

Previously, we determined the N-terminal amino acid sequences of a number of putative peribacteroid membrane proteins from soybean. Here, we report the cloning of a gene, GmN6L, that encodes one of these proteins. The protein encoded by GmN6L is similar in sequence to MtN6, an early nodulin expressed in Medicago truncatula roots in response to infection by Sinorhizobium meliloti. The GmN6L gene was strongly expressed in mature nodules but not in other plant organs. GmN6L protein was first detected 2 weeks after inoculation with Bradyrhizobium japonicum and was limited to the infected zone of nodules. GmN6L protein was found in symbiosomes isolated from mature soybean nodules, both as a soluble protein and as a peripheral membrane protein bound to the peribacteroid membrane. These data indicate that GmN6L is a late nodulin, which is not involved in the infection process. Homology between GmN6L and FluG, a protein involved in signaling in Aspergillus nidulans, suggests that GmN6L may play a role in communication between the host and microsymbionts during symbiotic nitrogen fixation.     

The role of sucrose synthase in the response of soybean nodules to drought

Experiments were carried out to investigate the effects of droughtstress on enzymatic activities related to carbon and nitrogenmetabolism in soybean nodules. Gradual drought stress, imposedby withholding water nutrients, resulted in declines in thewater potential of leaves and nodules consistent with a significantdecline in N2 fixation. However, the amounts of nitrogenasecomponents 1 and 2 were virtually unaffected by drought stress.Similarly, no significant changes could be detected in aspartateaminotransferase, phosphoenolpyruvate carboxylase, glutaminesynthetase or alkaline invertase activities throughout the experiment.In contrast, sucrose synthase (SS), one of the enzymes involvedin sucrose metabolism in legume nodules, declined dramaticallyin activity and in content within a few days of withholdingwater. Coincident with this decline in SS activity were significantincreases in the nodule contents of sucrose, total free aminoacids and ureides. The amounts of proline, however, did notincrease until some days later. It is suggested that SS mayplay a key role in the regulation of nodule carbon metabolismand, therefore, of nitrogen fixation under drought stress conditions. Key words: Glycine max, soybean, nodule metabolism, drought stress, sucrose synthase     

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

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

Soybean nodulin-26 gene encoding a channel protein is expressed only in the infected cells of nodules and is regulated differently in roots of homologous and heterologous plants.

Nodulin-26 (N-26) is a major peribacteroid membrane protein in soybean root nodules. The gene encoding this protein is a member of an ancient gene family conserved from bacteria to humans. N-26 is specifically expressed in root nodules, while its homolog, soybean putative channel protein, is expressed in vegetative parts of the plant, with its highest level in the root elongation zone. Analysis of the soybean N-26 gene showed that its four introns mark the boundaries between transmembrane domains and the surface peptides, suggesting that individual transmembrane domains encoded by a single exon act as functional units. The number and arrangement of introns between N-26 and its homologs differ, however. Promoter analysis of N-26 was conducted in both homologous and heterologous transgenic plants. The cis-acting elements of the N-26 gene are different from those of the other nodulin genes, and no nodule-specific cis-acting element was found in this gene. In transgenic nodules, the expression of N-26 was detected only in the infected cells; no activity was found in nodule parenchyma and uninfected cells of the symbiotic zone. The N-26 gene is expressed in root meristem of transgenic Lotus corniculatus and tobacco but not in untransformed and transgenic soybean roots, suggesting the possibility that this nodulin gene is controlled by a trans-negative regulatory mechanism in homologous plants. This study demonstrates how a preexisting gene in the root may have been recruited for symbiotic function and brought under nodule-specific developmental control.     

The aberrant cell walls of boron-deficient bean root nodules have no covalently bound hydroxyproline-/proline-rich proteins.

B-deficient bean (Phaseolus vulgaris L.) nodules examined by light microscopy showed dramatic anatomical changes, mainly in the parenchyma region. Western analysis of total nodule extracts examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one 116-kD polypeptide was recognized by antibodies raised against hydroxyproline-rich glycoproteins (HRGPs) from the soybean (Glycine max) seed coat. A protein with a comparable molecular mass of 116 kD was purified from the cell walls of soybean root nodules. The amino acid composition of this protein is similar to the early nodulin (ENOD2) gene. Immunoprecipitation of the soybean ENOD2 in vitro translation product showed that the soybean seed coat anti-HRGP antibodies recognized this early nodulin. Furthermore, we used these antibodies to localize the ENOD2 homolog in bean nodules. Immunocytochemistry revealed that in B-deficient nodules ENOD2 was absent in the walls of the nodule parenchyma. The absence of ENOD2 in B-deficient nodules was corroborated by performing hydroxyproline assays. Northern analysis showed that ENOD2 mRNA is present in B-deficient nodules; therefore, the accumulation of ENOD2 is not affected by B deficiency, but its assembly into the cell wall is. B-deficient nodules fix much less N2 than control nodules, probably because the nodule parenchyma is no longer an effective O2 barrier.     

The soybean NRAMP homologue,GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.     

The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean

Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N(2) fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N(2) fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro (33)P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance.     

Nodulin gene expression during soybean (Glycine max) nodule development

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod ⁺ fix^-mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod ⁺ fix^-mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.     

Soybean genotypic differences in sensitivity of symbiotic nitrogen fixation to soil dehydration

Nitrogen fixation activity by soybean (Glycine max (L.) Merr.) nodules has been shown to be especially sensitive to soil dehydration. Specifically, nitrogen fixation rates have been found to decrease in response to soil dehydration preceding alterations in plant gas exchange rates. The objective of this research was to investigate possible genetic variation in the sensitivity of soybean cultivars for nitrogen fixation rates in response to soil drying. Field tests showed substantial variation among cultivars with Jackson and CNS showing the least sensitivity in nitrogen accumulation to soil drying. Glasshouse experiments confirmed a large divergence among cultivars in the nitrogen fixation response to drought. Nitrogen fixation in Jackson was again found to be tolerant of soil drying, but the other five cultivars tested, including CNS, were found to be intolerant. Experiments with CNS which induced localized soil drying around the nodules did not result in decreases in nitrogen fixation rates, but rather nitrogen fixation responded to drying of the entire rooting volume. The osmotic potential of nodules was found to decrease markedly upon soil drying. However, the decrease in nodule osmotic potential occurred after significant decreases in nitrogen fixation rates had already been observed. Overall, the results of this study indicate that important genetic variations for sensitivity of nitrogen fixation to soil drying exist in soybean, and that the variation may be useful in physiology and breeding studies.     

Two classes of differentially regulated glutamine synthetase genes are expressed in the soybean nodule: a nodule-specific class and a constitutively expressed class

We have characterized two sets of cDNA clones representing the glutamine synthetase (GS) mRNA in soybean nodules. Using the 3-untranslated regions of a representative member of each set, as gene member(s) specific probes, we have shown that one set of the GS genes are expressed in a nodule-specific manner, while the other set is expressed in other tissues, besides the nodules. The nodule-specific GS genes are expressed in a developmentally regulated manner in the nodules, independent of the onset of nitrogen fixation. The other class of GS genes is expressed constitutively in all tissues tested, but its expression level is dramatically enhanced in nodules following onset of N2 fixation. The latter set of genes is also expressed in cotyledons of germinating seedlings in a developmentally regulated manner. Analysis of hybrid select translation products and genomic Southern blots suggests that multiple gene members in each class are expressed in the nodules.     

Characterization of the soybean early nodulin cDNA clone GmENOD55

Two cDNA clones of the soybean early nodulin GmENOD55 were characterized. These clones may represent two members of the soybean early nodulin gene family GmENOD55. GmENOD55 has an N-terminal signal peptide and it contains an internal domain consisting of proline and serine residues. Analyses of nodules lacking infection threads and intracellular bacteria suggest that the GmENOD55 gene is first expressed after release ofBradyrhizobium japonicum in plant cells. This conclusion is supported byin situ hybridization studies showing that the expression is restricted to the infected cell type.