Growth, nitrogen fixation and ammonium assimilation in common bean (Phaseolus vulgaris L) subjected to iron deficiency

A greenhouse experiment was carried out aiming to study the effect of iron deficiency on nitrogen fixation and ammonium assimilation in common bean nodules. Host-plant and nodule growth, symbiotic nitrogen fixation, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were analyzed in two common bean varieties subjected to iron deficiency. Results showed that host-plant and nodules growth, nitrogen fixation and GS activity decreased when under Fe-deficiency against an important increase of ammonium accumulation and GDH activity. Tolerant variety Flamingo is clearly less affected by iron deficiency than the sensitive one, Coco blanc. The allocation of iron to nodules and Fe use-efficiency for nodule growth and symbiotic nitrogen fixation were on the basis of the symbiotic performance of Flamingo under iron deprivation. Under Fe-deficiency, GDH take over GS the ammonium assimilation activity, particularly in the tolerant variety.     

Studies on the Relationship Between Photosynthesis and Nitrogen Fixation in the Symbiotic System of Soybean and Nodule Bacteria( Rhizobium)

The relationship between photosynthesis of soybean and nitrogen fixation of the nodules by symbiotic Rhizobium was studied. The contents of total nitrogen and chlorophyll, the net photosynthetic rate and seed yield of soybean were much higher in either hydroponically cultivated or field-grown plants inoculated with Rhizobium B16–11C (or Clark nodulating strain) than in control without inoculation (or Clark non-nodulating strain). These results show that the symbiotic nitrogen fixation has a beneficial effect on photosynthesis. However, the effect was indirect and slow so that there was no change in the net photosynthetic rate of the soybean leaves until three clays after removing nodules from the soybean roots. On the other hand, decreasing the photosynthate supply to nodule by shade, defoliation or shoot removal of the soybean, the nodule activity declined significantly. It seems that the supply of photosynthate to root nodule is a limiting factor for symbiotic nitrogen fixation. However, the diurnal variation of the nodule activity could not be explained by change neither in the contents of sucrose and starch of the root nodules nor in the ambient temperature. The factor controlling the diurnal variation deserves further study.     

大豆-根瘤菌共生体系光合与固氮关系研究

水培大豆和田间生长的大豆,接种根瘤菌 Rhizobium B16-11C 后植株全氮含量、叶片叶绿素含量和净光合速率及种子产量都明显增加。比较 Clark 大豆的结瘤品系和不结瘤品系获类似结果。摘除根瘤后3天内叶片净光合速率无明显变化。大豆植株遮阴、去叶或切掉地上部导致根瘤活性明显下降。但去豆荚不能提高根瘤固氮的比活性。根瘤活性的日变化不能用根瘤蔗糖、淀粉含量或周围温度的变化来解释,其控制因子尚待深入研究。     

Effectiveness of bacterization of soy plants by active races of nodule bacteria

Spontaneous nodule bacteria of soybean plants are absent from the Sutn-Eastern Kazakhstan soils. Therefore, bacterization of soybean plants with these bacteria is necessary for obtaining high crops of this culture. Among tested strains of soybean nodule bacteria, strain 623(a) was found to be the most effective one. The use of nitrogen with this strain stimulated the intensive formation of nodules, the growth and development of plants, the activation of physiological processes in them, and increased the yield of green mass, hay and grains as well as the content of protein in them and its yield per area unit. Mineral nitrogen added to soil at a concentration of 120 kg per hectare decreased the activity of symbiotic nitrogen fixation, increased the crop of green mass and hay, but decreased the yield of grain of soybean and other cultures sown after it in crop rotation.     

Effects of calcium deficiency on symbiotic nitrogen fixation

The mechanism of the effect of mild calcium deficiency on nitrogen metabolism of the symbiotic plant was studied from the distribution of calcium and of nitrogen and carbohydrate fractions in plant organs.

Nitrogen concentrations of all plant organs decreased with calcium deficiency. Addition of either a nitrogen or a calcium salt increased nitrogen concentrations. For roots as well as whole plants the effects of one salt were largely replaced by the other. These effects establish that calcium deficiency decreased the supply of fixed nitrogen from nodules to other organs. As weight of nodules was independent of calcium it follows that nodular efficiency was impaired.

Since nitrogen concentrations of nodules decreased rather than increased with calcium deficiency it is suggested that calcium deficiency interferes with the rate of reduction of nitrogen in the nodule rather than with the export of reduced nitrogen. Distribution of α-amino, amide, and ammonium nitrogen are consistent with this suggestion.

Nitrogen fixation was not limited by translocation of carbohydrates to nodules as calcium deficiency had little effect on the concentration of soluble carbohydrates and actually increased the concentration of starch in nodules.

Calcium deficiency depressed the calcium content of nodules so that nitrogen fixation may have been impaired by inadequate calcium for nodular structure or metabolism.

     

Metabolite profiles of nodulated alfalfa plants indicate that distinct stages of nodule organogenesis are accompanied by global physiological adaptations

An effective symbiosis between Sinorhizobium meliloti and its host plant Medicago sativa is dependent on a balanced physiological interaction enabling the microsymbiont to fix atmospheric nitrogen. Maintenance of the symbiotic interaction is regulated by still poorly understood control mechanisms. A first step toward a better understanding of nodule metabolism was the determination of characteristic metabolites for alfalfa root nodules. Furthermore, nodules arrested at different developmental stages were analyzed in order to address metabolic changes induced during the progression of nodule formation. Metabolite profiles of bacteroid-free pseudonodule extracts indicated that early nodule developmental processes are accompanied by photosynthate translocation but no massive organic acid formation. To determine metabolic adaptations induced by the presence of nonfixing bacteroids, nodules induced by mutant S. meliloti strains lacking the nitrogenase protein were analyzed. The bacteroids are unable to provide ammonium to the host plant, which is metabolically reflected by reduced levels of characteristic amino acids involved in ammonium fixation. Elevated levels of starch and sugars in Fix(-) nodules provide strong evidence that plant sanctions preventing a transformation from a symbiotic to a potentially parasitic interaction are not strictly realized via photosynthate supply. Instead, metabolic and gene expression data indicate that alfalfa plants react to nitrogen-fixation-deficient bacteroids with a decreased organic acid synthesis and an early induction of senescence. Noneffective symbiotic interactions resulting from plants nodulated by mutant rhizobia also are reflected in characteristic metabolic changes in leaves. These are typical for nitrogen deficiency, but also highlight metabolites potentially involved in sensing the N status.     

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.     

Evidence that P deficiency induces N feedback regulation of symbiotic N2 fixation in white clover (Trifolium repens L.)

Trifolium repens L. was grown to test the following hypotheses: when P is deficient (i) N2 fixation decreases as a result of the plant's adaptation to the low N demand, regulated by an N feedback mechanism, and (ii) the decrease in the photosynthetic capacity of the leaves does not limit N2 fixation. Severe P deficiency prevented nodulation or stopped nodule growth when the P deficiency occurred after the plants had formed nodules. At low P, the proportion of whole-plant-N derived from symbiotic N2 fixation decreased, whereas specific N2 fixation increased and compensated partially for poor nodulation. Leaf photosynthesis was reduced under P deficiency due to low Vc,max and Jmax. Poor growth or poor performance of the nodules was not due to C limitation, because (i) the improved photosynthetic performance at elevated pCO2 had no effect on the growth and functioning of the nodules, (ii) starch accumulated in the leaves, particularly under elevated pCO2, and (iii) the concentration of WSC in the nodules was highest under P deficiency. Under severe P deficiency, the concentrations of whole-plant-N and leaf-N were the highest, indicating that the assimilation of N exceeded the amount of N required by the plant for growth. This was clearly demonstrated by a strong increase in asparagine concentrations in the roots and nodules under low P supply. This indicates that nodulation and the proportion of N derived from symbiotic N2 fixation are down-regulated by an N feedback mechanism.     

Soybean ureide transporters play a critical role in nodule development,function and nitrogen export

Legumes can access atmospheric nitrogen through a symbiotic relationship with nitrogen‐fixing bacteroids that reside in root nodules. In soybean, the products of fixation are the ureides allantoin and allantoic acid, which are also the dominant long‐distance transport forms of nitrogen from nodules to the shoot. Movement of nitrogen assimilates out of the nodules occurs via the nodule vasculature; however, the molecular mechanisms for ureide export and the importance of nitrogen transport processes for nodule physiology have not been resolved. Here, we demonstrate the function of two soybean proteins – GmUPS1‐1 (XP_003516366) and GmUPS1‐2 (XP_003518768) – in allantoin and allantoic acid transport out of the nodule. Localization studies revealed the presence of both transporters in the plasma membrane, and expression in nodule cortex cells and vascular endodermis. Functional analysis in soybean showed that repression of GmUPS1‐1 and GmUPS1‐2 in nodules leads to an accumulation of ureides and decreased nitrogen partitioning to roots and shoot. It was further demonstrated that nodule development, nitrogen fixation and nodule metabolism were negatively affected in RNAi UPS1 plants. Together, we conclude that export of ureides from nodules is mediated by UPS1 proteins, and that activity of the transporters is not only essential for shoot nitrogen supply but also for nodule development and function.     

Growth, nitrogen fixation and ammonium assimilation in common bean (Phaseolus vulgaris): effect of phosphorus

The impact of phosphorous nutrition on plant growth, symbiotic N₂ fixation, ammonium assimilation, carbohydrate and amino-acid accumulation, as well as on nitrogen, phosphorus and ATP content in tissues in common bean ( Phaseolus vulgaris ) plants was investigated. Plants inoculated with Rhizobium tropici CIAT899 were grown in Leonard jars under controlled conditions, with P-deficient (0 and 0.1 m M ), P-medium (0.5, 1 and 1.5 m M ) and P-high (2 m M ) conditions in a N-free nutrient solution. The P application, increased leaf area, whole plant DW (67%), nodule biomass (4-fold), and shoot and root P content (4- and 6-fold, respectively) in plant harvested at the onset of flowering (28-days-old). However, P treatments decreased the total soluble sugar and amino acid content in vegetative organs (leaf, root and nodules). The root growth proved less sensitive to P deficiency than did shoot growth, and the leaf area was significantly reduced at low P-application. The absence of a relationship between shoot N content, and P levels in the growth medium could indicate that nitrogen fixation requires more P than does plant growth. The optimal amount for the P. vulgaris – R. tropici CIAT899 symbiosis was 1.5 m M P, this treatment augmented nodule-ARA 20-fold, and ARA per plant 70-fold compared with plants without P application.     

Recovery of nodulated cowpea plants (Vigna unguiculata (L.) Walp.) from waterlogging during vegetative growth

Summary Waterlogging pot-grown cowpea plants for eight days (24–32 from sowing) during the period of rapid vegetative growth when symbiotic nitrogen fixation rates are also increasing at maximum rate had effects on anatomical, morphological and physiological attributes of the legume system viz reduced nodule production and increased nodule cortication, stimulated adventitious root formation at, or near, the water table but killed older submerged roots and nodules, caused the main stem to become hypertrophic just above the water line, accelerated senescence of the lowermost leaves and delayed branch formation. After re-draining the rooting medium nodule growth and symbiotic fixation activity occurred at the expense of rapid recovery by the host plant. The physiological basis for these findings in relation to identifying cowpea cultivars more tolerant of waterlogged soils is discussed.One of a series of papers describing work undertaken in a collaborative project with the International Institute of Tropical Agriculture, Nigeria, sponsored by the U.K. Ministry of Overseas Development.     

The Physiological Mechanisms Underlying N₂-Fixing Common Bean (<i>Phaseolus vulgaris</i> L.) Tolerance to Iron Deficiency

Iron is an essential element for plants as well as all living organisms, functioning in various physiological and biochemical processes such as photosynthesis, respiration, DNA synthesis, and N₂ fixation. In the soil, Fe bioavailability is extremely low, especially under aerobic conditions and at high pH ranges. In contrast, plants with nodules on their roots that fix atmospheric nitrogen need much more iron. To highlight the physiological traits underlying the tolerance of N₂-fixing common bean to iron deficiency, two genotypes were hydroponically cultivated in a greenhouse: Coco nain (CN) and Coco blanc (CB). Plants were inoculated with an efficient strain of Rhizobium tropici, CIAT899, and received a nutrient solution added with 0 µM Fe (severe Fe deficiency, SFeD), 5 µM Fe (moderate Fe deficiency, MFeD) or 45 µM Fe (control, C). Several physiological parameters related to photosynthesis and symbiotic nitrogen fixation were then analyzed. Iron deficiency significantly reduced whole plant and nodule growth, chlorophyll biosynthesis, photosynthesis, leghemoglobin (LgHb), nitrogenase (N₂ase) activity, nitrogen, and Fe nutrition, with some genotypic differences. As compared to CB, CN maintained better Fe allocation to shoots and nodules, allowing it to preserve the integrity of its photosynthetic and symbiotic apparatus, thus maintaining the key functional traits of the plant metabolism (chlorophyll biosynthesis and photosynthesis in shoots, leghemoglobin accumulation, and nitrogenase activity in root nodules). Plant growth depends on photosynthesis, which needs to be supplied with sufficient iron and nitrogen. Fe deficiency stress index (FeD-SI) and Fe use efficiency (FeUE) are two physiological traits of tolerance that discriminated the studied genotypes.     

A small heat shock protein,GmHSP17.9, from nodule confers symbiotic nitrogen fixation and seed yield in soybean

Legume–rhizobia symbiosis enables biological nitrogen fixation to improve crop production for sustainable agriculture. Small heat shock proteins (sHSPs) are involved in multiple environmental stresses and plant development processes. However, the role of sHSPs in nodule development in soybean remains largely unknown. In the present study, we identified a nodule-localized sHSP, called GmHSP17.9, in soybean, which was markedly up-regulated during nodule development. GmHSP17.9 was specifically expressed in the infected regions of the nodules. GmHSP17.9 overexpression and RNAi in transgenic composite plants and loss of function in CRISPR-Cas9 gene-editing mutant plants in soybean resulted in remarkable alterations in nodule number, nodule fresh weight, nitrogenase activity, contents of poly β-hydroxybutyrate bodies (PHBs), ureide and total nitrogen content, which caused significant changes in plant growth and seed yield. GmHSP17.9 was also found to act as a chaperone for its interacting partner, GmNOD100, a sucrose synthase in soybean nodules which was also preferentially expressed in the infected zone of nodules, similar to GmHSP17.9. Functional analysis of GmNOD100 in composite transgenic plants revealed that GmNOD100 played an essential role in soybean nodulation. The hsp17.9 lines showed markedly more reduced sucrose synthase activity, lower contents of UDP-glucose and acetyl coenzyme A (acetyl-CoA), and decreased activity of succinic dehydrogenase (SDH) in the tricarboxylic acid (TCA) cycle in nodules due to the missing interaction with GmNOD100. Our findings reveal an important role and an unprecedented molecular mechanism of sHSPs in nodule development and nitrogen fixation in soybean.     

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.     

Phosphorus deprivation affects composition and spatial distribution of membrane lipids in legume nodules

In legumes, symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. In a nodule, N-fixing rhizobia are surrounded by symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N₂-fixing legumes have a higher requirement for P than legumes grown on mineral N. As in the previous studies, in P deficiency, barrel medic (Medicago truncatula) plants had impaired SNF activity, reduced growth, and accumulated less phosphate in leaves, roots, and nodules compared with the plants grown in P sufficient conditions. Membrane lipids in M. truncatula tissues were assessed using electrospray ionization–mass spectrometry. Galactolipids were found to increase in P deficiency, with declines in phospholipids (PL), especially in leaves. Lower PL losses were found in roots and nodules. Subsequently, matrix-assisted laser desorption/ionization–mass spectrometry imaging was used to spatially map the distribution of the positively charged phosphatidylcholine (PC) species in nodules in both P-replete and P-deficient conditions. Our results reveal heterogeneous distribution of several PC species in nodules, with homogeneous distribution of other PC classes. In P poor conditions, some PC species distributions were observed to change. The results suggest that specific PC species may be differentially important in diverse nodule zones and cell types, and that membrane lipid remodeling during P stress is not uniform across the nodule.

ESI–MS and matrix-assisted laser desorption ionization–mass spectrometry imaging reveal alterations in Medicago truncatula nodules membrane lipid composition and spatial distribution in phosphorus deficiency.     

Catalase Activity of Nodules in the Ureide- and Amide-transporting Legume Plants

The relations of catalase activity to the efficiency of symbiotic dinitrogen fixation and leghemoglobin (Lb) content were investigated in roots and nodules of several legume plant species together with the catalase distribution between the inner bacteroidal and the outer cortical nodule tissues. The catalase activity in the nodules exceeded that of the roots of the amide- and ureide-synthesizing plant species by one and two orders of magnitude. During the growth period, catalase activity and Lb content changed in parallel and reached their highest levels early in the stage of flowering or fruit formation, depending on plant species. In the case of effective symbiosis, catalase activity in the nodules was 2.5–5 times higher than in the case of ineffective symbiosis. Catalase activity in the bacteroidal zone of the nodules was several times higher than that of the cortical tissue, and two nodule tissues differed in catalase activity more notably in the plant species exporting ureides. The authors suggest that high catalase activity in the nodules, especially in their bacteroidal zone, is essential for the efficient functioning of the symbiotic system of dinitrogen fixation in both ureide- and amide-transporting plants.     

Isocitrate dehydrogenase of Bradyrhizobium japonicum is not required for symbiotic nitrogen fixation with soybean

A mutant strain of Bradyrhizobium japonicum USDA110 lacking isocitrate dehydrogenase activity was created to determine whether this enzyme was required for symbiotic nitrogen fixation with soybean (Glycine max cv. Williams 82). The isocitrate dehydrogenase mutant, strain 5051, was constructed by insertion of a streptomycin resistance gene cassette. The mutant was devoid of isocitrate dehydrogenase activity and of immunologically detectable protein, indicating there is only one copy in the genome. Strain 5051 grew well on a variety of carbon sources, including arabinose, pyruvate, succinate, and malate, but, unlike many microorganisms, was a glutamate auxotroph. Although the formation of nodules was slightly delayed, the mutant was able to form nodules on soybean and reduce atmospheric dinitrogen as well as the wild type, indicating that the plant was able to supply sufficient glutamate to permit infection. Combined with the results of other citric acid cycle mutants, these results suggest a role for the citric acid cycle in the infection and colonization stage of nodule development but not in the actual fixation of atmospheric dinitrogen.     

Regulation of soybean nitrogen fixation in response to rhizosphere oxygen: I. Role of nodule respiration

Nitrogen fixation (acetylene reduction) rates of nodules on intact field-grown soybean (Glycine max) subjected to altered oxygen concentration (0.06-0.4 cubic millimeter per cubic millimeter) returned to initial rates during an 8-hour transitory period. Hydroponically grown soybean plants also displayed a transitory (1-4 hours) response to changes in the rhizosphere oxygen concentration after which the fixation rates returned to those observed under ambient oxygen concentrations. It was hypothesized that soybean nodules contain a regulatory mechanism which maintains a stable oxygen concentration inside nodules at a sufficiently low concentration to allow nitrogenase to function. A possible physiological mechanism which could account for this regulation is adjustment in nodule respiration activity such that nodule oxygen concentration and nitrogen fixation are maintained at stable levels. Experiments designed to characterize the non-steady-state oxygen response and to test for the presence of nodule respiratory control are presented. Non-steady-state acetylene reduction and nodule respiration (oxygen uptake) rates measured after alterations in the external oxygen concentration indicated that the regulatory mechanism required 1 to 4 hours to completely adjust to changes in the external oxygen concentration. Steady-state nodule respiration, however, did not respond to alterations in the rhizosphere oxygen concentration. It was concluded that soybean nodules can adjust to a wide range of rhizosphere oxygen concentrations, but the mechanism which controls nitrogen fixation rates does not involve changes in the nodule respiration rate.