Plant-endophyte symbiosis in non-leguminous plants

Summary A wide taxonomic range of non-leguminous dicotyledonous plants bear root nodules and are able to fix atmospheric nitrogen. These plants belong to the orders Casuarinales, Myricales, Fagales, Rhamnales, Coriariales, and Rosales. Actinomycetes are involved in the root-nodule symbiosis. Nitrogen fixation is inhibited by hydrogen and carbon monoxide. Combined nitrogen depress nodule formation, but nitrogen fixation still occurs in the presence of combined nitrogen in the medium. In nitrogen-free medium Alnus plants fix in one season of 48 weeks 500 mg N per plant and Ceanothus plants 760 mg N per plant. Fixation by the other plant species is about of the same order. Field estimates showed that the nitrogen increase of the soil was about 61.5–157 kg N per ha per annum, depending on the age of the trees, under Alnus, 58.5 kg N per ha per annum under Casuarina, about 60 kg N per ha per annum under Ceanothus, 27–179 kg N per ha per annum underHippopha? rhamnoides, and about 61.5 kg N per ha per annum underDryas drummondii with someShepherdia spp. Non-leguminous root nodules belong to two types: coralloid root nodules and root nodules where the apex of each nodule lobe produces a negatively geotropic root. The primary infection occurs through the root hairs where a curling effect is observed. In the host cells the endophyte presents itself in three forms: hyphae, vesicles and bacteria-like cells. Vesicles are probably associated with nitrogen fixation, whereas the bacteria-like cells function in the endophyte's survival and dispersal. The endophyte is an obligate symbiont. TheAlnus glutinosa endophyte has been isolated and grownin vitro in root-nodule callus tissue. However, the isolated endophyte produces only ineffective root nodules in re-inoculation tests.     

Free-Living Rhizobium Strain Able To Grow on N2 as the Sole Nitrogen Source

A Rhizobium strain isolated from stem nodules of the legume Sesbania rostrata was shown to grow on atmospheric nitrogen (N₂) as the sole nitrogen source. Non-N₂-fixing mutants isolated directly on agar plates formed nodules that did not fix N₂ when inoculated into the host plant.     

A mutant of the cyanobacterium Anabaena variabilis ATCC 29413 lacking cyanophycin synthetase: growth properties and ultrastructural aspects

The gene cphA encoding cyanophycin synthetase was interrupted in Anabaena variabilis ATCC 29413 by insertional mutagenesis. The mutant lacked cyanophycin granules and the polar nodules of heterocysts. The mutant grew as fast as the wild-type irrespective of the nitrogen source at low light intensity whereas growth on N(2) was somewhat reduced in high light. It is concluded that cyanophycin metabolism and polar nodules are not essential for aerobic N(2) fixation.     

Organic acid accumulation may inhibit N2 fixation in phosphorus-stressed lupin nodules.

Nodulated lupins (Lupinus angustifolius cv. Wonga) were hydroponically grown under conditions of low phosphate (LP) or adequate phosphate (HP) to assess the effect of phosphoenolpyruvate carboxylase (PEPC)-derived organic acids on nitrogen assimilation in LP nodules. LP conditions are linked to altered organic acid metabolism, by the engagement of PEP metabolism via PEPC. In LP nodules, the enhanced organic acid synthesis may reduce the available organic carbon for nitrogen assimilation. The diversion of carbon between the organic acid- and amino acid pools was assessed through key nodular enzymes and (14)CO(2) metabolism. Under LP conditions, increased rates of organic acid synthesis via PEPC and malate dehydrogenase (MDH), coincided with reduced nitrogen assimilation via aspartate aminotransferase (AAT), aspartate synthetase (AS) and glutamine synthetase (GS)/glutamate synthase (GOGAT) activities. There was a preferential metabolism of nodular (14)CO(2) into organic acids and particularly into malate. High malate levels were associated with reduced N(2) fixation and synthesis of amino acids. These results indicate that phosphorus deficiency can enhance malate synthesis in nodules, but that excessive malate accumulation may inhibit N(2) fixation and nitrogen assimilation.     

Stem Nodules in Aeschynomene indica and Their Capacity of Nitrogen Fixation

There has been no report on stem nodules with nitrogen fixing activity. Aeschynomene indica produce nodules on the aerial parts, and the stem nodules proved to be capable of considerable nitrogen fixation [C₂H₄ produced c. 4 μmol (g fresh weight)^-1 h^-1]. The stem nodules embed reddish tissues, and a rod-shaped bacterium was isolated from the tissues. The bacterium was ascertained to form root and stem nodules on seedlings of A. indica.     

大叶相思的结瘤固氮和吸氢酶活性

本文报道大叶相思的结瘤固氮和氢酶活性的研究结果。大叶相思结瘤状况与其他含羞草亚科的树种相似,刚形成的幼瘤为单生球状或椭圆形,以后顶端伸长或分叉,呈分叉状、姜状和扇状。同一植株的不同成熟度根瘤的固氮活性也不同,成熟壮瘤固氮活性最高,幼瘤次之,衰老瘤最低。不同立地条件下种植的大叶相思根瘤固氮活性虽有差异,但生长在pH4.7的酸性红壤中的大叶相思,根瘤固氮活性仍具有较高水平。大叶相思根瘤固氮活性也有明显的季节变化,夏秋较高,春冬较低。根瘤离体后7小时内固氮活性变化不大,甚至在离体后21小时内仍维持一定水平的固氮活性。大叶相思根瘤具有吸氢酶,其吸H_2活性在最初3小时内随时间延长而升高,3—7小时内仍维持一定水平。在根瘤固氮系统中注入外源分子H_2,可提高固氮酶活性,外源H_2最适浓度为7.5％。由于其根瘤具有催化吸收分子H_2的氢酶系统,能吸收利用固氮反应所放出的大量H_2,因而能更有效地利用光合产物于固氮过程。大叶相思根瘤离体后能较长时间维持高的固氮活性水平,可能与其吸H_2酶系统有关。     

Nitrogen Fixation and Allantoin Formation in Soybean Plants

The activity of nitrogenase and the concentration of ammonia and allantoin (+ allantoic acid) in root nodules were measured throughout the growth period of soybean plants. Nitrogenase activity measured by acetylene reduction increased with plant growth and reached a maximum level at the flowering period. The level of ammonia and allantoin concentration in nodules was parallel with increased nitrogenase activity. At the late reproductive stage (pod-forming period), nitrogenase activity showed a marked decrease, but the ammonia and allantoin in the nodules remained at a constant level. Detached nodules from 56 day-old soybean plants were exposed to ¹⁵N₂ gas, and the distribution of ¹⁵N among nitrogen compounds was investigated. Enrichment of ¹⁵N in allantoin and allantoic acid reached a fairly high level after 90 min of nitrogen fixation; ca. 22% of ¹⁵N in acid-soluble nitrogen compounds was incorporated into allantoin + allantoic acid. In contrast, enrichment of ¹⁵N in amide nitrogen was relatively low. No significant ¹⁵N was detected in the RNA fraction. The data suggested that ureide formation in nitrogen-fixing root nodules did not take place through the breakdown of nucleic acids, but directly associated with the assimilating system of biologically fixed nitrogen.     

Genetics of promiscuous nodulation in soybean: nodule dry weight and leaf color score

The symbiotic relationship between the soybean plant and rhizobium results in fixation of atmospheric nitrogen (N(2)) in the root nodules, with the result that nitrogenous fertilization of the soybean is unnecessary. The effectiveness of nodule formation and N(2) fixation with rhizobial strains is under genetic control with two general categories identified: (1) promiscuous, which produces functional nodules with cowpea-type rhizobial strains; and (2) nonpromiscuous, which forms no or nonfunctional nodules with these strains. The segregation pattern of this promiscuity trait was studied using nodule dry weight (NDW) and leaf color score (LCS) as indicators of N(2) fixation effectiveness. Individual plants in each of six populations [P(1) = nonpromiscuous, P(2) = promiscuous, F(1) = P(1) x P(2) (and the reciprocal cross), BC(1)(P(1)) = F(1) (female) x P(1), BC(1)(P(2)) = F(1) (female) x P(2), F(2)] were scored for these characters after inoculation with a rhizobial strain that would distinguish between both types. For NDW, nonpromiscuity was found to be partially dominant (h/d = 0.37), controlled by four loci. For LCS, nonpromiscuity was shown to be almost completely dominant (h/d = 0.74), controlled by two loci. LCS was a more meaningful estimate of N(2) fixation because it represented the total effectiveness of nodulation to provide nitrogen for the plant.     

Nitrogen fixation (C2H2 reduction) by Medicago nodules and bacteroids under sodium chloride stress

Medicago ciliaris (L.) All., a salt-tolerant legume, was not nodulated by Rhizobium meliloti (2011), a strain commonly used for field inoculation of alfalfas. A strain of Rhizobium meliloti (ABS7) was isolated from saline Algerian soils. It is generally more salt-resistant than strain 2011, exhibits a higher rate of growth and induces the formation of nodules on M. ciliaris . C₂H₂ reduction activity of M. ciliaris nodules was inhibited by 50% in the presence of 200 m M NaCl in the culture medium. whereas 100 m M NaCl was sufficient to inhibit the activity of nodules of M. sativa (L. cv. Europe). C₂H₂ reduction by bacteroids, isolated from nodules of the two species of alfalfa, was directly inhibited by the presence of NaCl in the incubation medium. In both cases, glucose could support bacteroid nitrogen fixation, but only in a narrow range of O₂ tensions. Bacteriods from M. ciliaris were more tolerant to salt than M. sativa ones. The salt resistance of bacteroids from nodules of plants watered with NaCl solutions was not improved in either species. Salt directly added to the incubation mixture of bacteroids or to the culture medium of plants inhibited O₂ uptake of bacteroids isolated from nodules of both M. ciliaris and M. sativa . The depressive effect of NaCl on bacteroid C₂H₂ reduction could be directly related to the drop in bacteroid respiration. The nitrogen fixation capacity of the M. ciliaris-Rhizobium meliloti (ABS7) symbiosis under saline conditions leads us to recommend the introduction of this association in salt-troubled areas.     

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.     

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.     

Carbon Metabolism, Nitrogen Assimilation, and Seed Yield of Cowpea (Vigna unquiculata L. Walp.) Grown in an Adverse Temperature Regime

When grown in an environment known not to favour the productionof large seed yields (warm days-cool nights; 33–19 °C),non-nodulated plants of cowpea cv. K 2809 supplied with abundantinorganic nitrogen not only assimilated N more rapidly but alsoproduced larger total dry weights and seed yields than plantsdependent on Rhizobium CB 756. Remobilization of nitrogen fromvegetative organs started sooner in nitrate-dependent than innodulated plants and contributed 69 and 47%, respectively, tothe N content of mature fruits. Plants dependent on nodulesrelied more on current assimilation of nitrogen during the laterstages of fruit growth than those given inorganic N; they alsoutilized a larger proportion of shoot-derived photosynthatesin growth of organs below ground and in the respiratory activitiesof both nodules and supporting roots. Although nitrate-dependentplants developed larger shoot systems than those relying onnodules, the distribution of carbon and nitrogen to leaves decreasedmarkedly as branches extended during early reproductive growth.The respiration of roots on nodulated plants became more efficientduring the later stages of fruit growth whereas the populationof secondary nodules present at this stage of development respiredless efficiently (mg C consumed per mg N assimilated) than theprimary nodules present earlier during development.     

Response of soybean-Rhizobium symbioses to mineral nitrogen

Summary The effect of mineral nitrogen on establishment and activity of symbioses between soybean and several strains ofRhizobium japonicum and on the establishment of nodules ofR. japonicum isolated from nodules of field crops is studied. All strains were highly susceptible to the effects of 200 ppm NO₃–N on the establishment of symbiosis; 50 ppm NO₃–N had little effect. Response of symbioses establishhed in the absence of mineral N to short term exposure to nitrate or ammonium varied significantly between strains. Nodule isolates from soybean crops growing in nitrifying soil were no less susceptible to the inhibitory effects of mineral N on nodule formation than a laboratory culture of the commercial inoculant strain.     

Effects of drought on nitrogen fixation in soybean root nodules

Soybean plants [Glycine max (L.) Merr.] were grown in silica sand and were drought stressed for a 4 week period during reproductive development and without any mineral N supply in order to maximize demand for fixed nitrogen. A strain of Bradyrhizobium japonicum that forms large quantities of polysaccharide in nodules was used to determine whether or not the supply of reduced carbon to bacteroids limits nitrogenase activity. A depression of 30–40% in nitrogen content in leaves and pods of stressed plants indicated a marked decline in nitrogen fixation activity during the drought period. A 50% increase in the accumulation of bacterial polysaccharide in nodules accompanied this major decrease in nitrogen fixation activity and this result indicates that the negative impact of drought on nodules was not due to a depression of carbon supply to bacteroids. The drought treatment resulted in a statistically significant increase in N concentration in leaves and pods. Because N concentration and chlorophyll concentration in leaves were not depressed, there was no evidence of nitrogen deficiency in drought‐stressed plants, and this result indicates that the negative impact of drought on nodule function was not the cause of the depression of shoot growth. At the end of the drought period, the concentration of carbohydrates, amino nitrogen, and ureides was significantly increased in nodules on drought‐stressed plants. The overall results support the view that, under drought conditions, nitrogen fixation activity in nodules was depressed because demand for fixed N to support growth was lower.     

Plant symbiotic mutants as a tool to analyse nitrogen nutrition and yield relationship in field-growth peas (Pisum sativum L.)

Pisum sativum L. is known for high seed and protein yields but also for.yield instability. Because legumes utilize two sources of nitrogen (atmospheric N₂ fixed in nodules and assimilation of soil mineral N), studies on their nitrogen nutrition is more complex than in other plants. In this work, pea symbiotic mutants (with no nodules at all ([Nod^-]), with inefficient nodules ([Nod⁺Fix^-]) or showing an hypernodulating and a ‘nitrate-tolerant symbiosis’ character ([Nod⁺⁺Nts]), their semi-leafless isogenic homologues and the parental control line cv Frisson were fertilized with three levels of mineral nitrogen (0, 25 or 50 g N m^-2) to generate a range of mineral nitrogen regimes in the same genetic background. Impact of the source and level of nitrogen nutrition was measured on reproductive development, growth, nitrogen accumulation and seed yield. It was shown that a N deficiency induced flowering termination. It also led to a large decrease in the number of seeds produced and the amount of N accumulated in forage and in seeds, when little effect was observed on the progression rates of reproductive stages along the stem. The single seed weight and the amount of dry matter accumulated in forage neither responded strongly to N deficiency. The source of nitrogen was shown to be of little importance to yield but the application of about 50 g N m^-2 was necessary to reach the yield of the control cv Frisson when exclusive assimilation was ensuring the N requirements of the plant. Despite the fact that the nitrate-tolerant and hypernodulating mutant P64 used in this study did not yield as well as the parent cv Frisson, it is proposed that [Nod⁺⁺Nts] characters could act as a yield regulating factor.     

Isolation of an Asm− mutant of Rhizobium japonicum defective in symbiotic N2-fixation

Abstract A mutant strain of Rhizobium japonicum (CJ9) unable to assimilate ammonium (Asm⁻) was isolated following mutagenesis with N -methyl N -nitro-nitrosoguanidine (NTG). Glutamate synthase activity was not detectable in cell-free extracts of the mutant strain in contrast to the wild type and revertant strains. Although mutant CJ9 induced nitrogenase activity in an 'in vitro' assay system under microaerobic conditions, it failed to fix nitrogen (acetylene reduction) in soybean root nodules. These properties of mutant CJ9 constitute a new Asm⁻ mutant class in Rhizobium spp.     

Rhizobial factors required for stem nodule maturation and maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.     

The nodulation of micro-propagated plants of Parasponia andersonii by tropical legume rhizobia

A simple clonal micro-propagation system for Parasponia andersoniiwas employed to study the nodulation response of this non-legumeto inoculation by the broad host range Rhizobium sp. NGR234,isolated from Lablab purpureus, and also to tropical legumerhizobia isolated from Aeschynomene species. Partially effectivenodules, assayed by acetylene reduction and ¹⁵N dilution procedures,were induced with strain NGR234 and its spontaneous streptomycinresistantmutant ANU240. Effective nodules were produced by one of theAeschynomene strains (ORS302) tested, with rates of acetylenereduction comparable to those of root nodules produced by Bradyrhizobiumstrain CP279, originally isolated from P. andersonii. Lightand transmission electron microscopy showed that there was acorrelation between the nitrogen fixing capability of the symbiosisbetween NGR234 and Parasponia and the number of persistent infection(fixation) threads within the nodule cells. Key words: Parasponia, Bradyrhizobium, Rhizobium, Aeschynomene, micro-propagation, root nodules, nitrogen fixation     

Formation of Nitrosylleghemoglobin in Nodules of Nitrate-Treated Cowpea and Pea Plants

The formation of nitrosylleghemoglobin (LbNO) was examined incowpea and pea nodules in relation to the inhibition of nitrogenfixation by nitrate. Leghemoglobin was of the ferrous type andwas mainly converted to LbNO in cowpea nodules when the acetylene-reducingactivity decreased to 45% of control values as a result of thesupply of nitrate. In nodules of nitrate-treated pea plants,leghemoglobin was also of the ferrous type and LbNO was a minorcomponent of leghemoglobin. The levels of LbNO isolated fromnodules corresponded to the levels of LbNO calculated from equilibriumconstants for LbNO and the concentration of nitrite in nodules.The dissociation rate constants for LbNO from both cowpea andpea were much smaller than those for LbO₂ or LbCO, as is alsothe case in soybean. These results indicate that the inhibition of the functionsof leghemoglobin, due to the accumulation of LbNO, induces adecrease in nitrogen fixation in cowpea nodules, and that theinhibition of nitrogen fixation in pea nodules is not relatedto the formation of LbNO. (Received July 2, 1990; Accepted October 9, 1990)