Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

Correlations between the ascorbate-glutathione pathway and effectiveness in legume root nodules

Legume root nodules use the ascorbate-glutathione pathway to remove harmful H₂O₂. In the present study. effective and ineffective nodules from soybean and alfalfa were compared with regard to this pathway. Effective nodules had higher activity of all 4 enzymes (ascorbate peroxidase, EC 1. 11. 1. 11: monodehydroascorbate reductase, EC 1. 6. 5. 4: dehydroascorbate reductase, EC 1. 8. 5. 1: and glutathione reductase, EC 1. 6. 4. 2). The concentration of thiol tripeptides (primarily homoglutathione) was about 1 m M in effective nodules – a level 3–4-fold higher than in ineffective nodules. Effective nodules contained higher levels of NAD⁺. NADP⁺ and NADPH. but not of NADH or ascorbate. The increased capacity for peroxide scavenging in effective nodules as compared to ineffective nodules emphasizes the important protective role that this pathway may play in processes related to nitrogen fixation.     

外源葡萄糖对山定子生长及根系氮素代谢的影响

以有机质含量仅为0.65%的低碳冲积沙土为栽培基质,以当年生山定子幼苗为试材,分别添加与土壤本体微生物生物量碳(MBC)等量的碳量(2 g·kg^-1)、5倍MBC碳量(10g·kg^-1)的葡萄糖,以不添加葡萄糖为对照,处理后0~30 d内定期采集根系样品,研究外源葡萄糖对低碳土壤中山定子幼苗生长、根系构型及氮素代谢的影响.结果表明:5倍MBC碳源处理后山定子幼苗的株高、总生物量、总根长和根表面积分别显著增加12.3%、26.4%、23.2%和14.6%,而茎粗、根体积和平均直径无显著变化.等量及5倍MBC碳源处理均显著提高了山定子的根系活力,分别在第3和15天达到峰值,高于对照119.1%和75.7%.在整个处理期间,等量及5倍MBC碳源处理显著增加了根中NO_3^-、NO_2^-和NH_4^+含量;整体上,等量及5倍MBC碳源处理均显著增强根系中硝酸还原酶、谷氨酰胺合酶、谷氨酸脱氢酶、谷氨酸合酶、谷草转氨酶和谷丙转氨酶的活性,其中5倍MBC处理的作用最显著.5倍MBC的外源葡萄糖浓度更有利于促进低碳土壤中山定子根系中氮素的吸收代谢过程,诱导植株生长、干物质积累和根系构型改变.     

Enzymes of ammonia assimilation in root nodules of Trigonella foenum-graecum

The main pathway of ammonia assimilation in the root nodules of Trigonella foenum-graecum is via nodule cytosol glutamine synthetase-glutamate synthase.     

Subcellular localization of enzymes involved in the assimilation of Ammonia by soybean root nodules

Ammonia, the primary product of nitrogen fixation is rapidly incorporated into a number of amino acids such as glutamate and aspartate. A novel enzyme system glutamine: 2-oxoglutarate aminotransferase oxidoreductase, which probably has an important role in ammonia assimilation has been detected, in the present studies, in the rhizobial fraction of soybean root nodules and in Rhizobium japonicum grown in culture. The role of this latter enzyme and other enzymes such as glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in ammonia assimilation by soybean nodules is discussed.     

Extracellular invertase of Rhizobium japonicum and its role in free sugar metabolism in the developing root nodules of Sesbania grandiflora

Extracellular invertase of Rhizobium japonicum and its role in free sugar metabolism in the developing root nodules of Sesbania grandiflora L. was studied. The enzyme hydrolysed sucrose extracellularly, and its release was substrate inducible. 0.1 Mβ-mercaptoethanol released the cell-bound form of this enzyme. The production of invertase was low when glucose, galactose, mannose, fructose and raffinose were used as carbon sources in the growth medium. In the developing nodules sucrose was the major sugar. The content of fructose was low in comparison with that of glucose – suggesting that in the nodules, fructose is converted to glucose prior to its entry into the bacterial cell. The content of glucose synchronised with the pattern of change in the activity of invertase in the nodules.     

Aldrin epoxidase from soybean root nodules

Soybean (Glycine max cv Forrest) root nodule homogenates oxidized aldrin to its epoxide, dieldrin. In crude tissue brei, addition of an NADPH-generating system was inhibitory to epoxidation. However, anaerobic gel filtration and sucrose density separation removed factors required for inhibition by NADPH, allowing a normal stimulation by the NADPH-generating system. In fractions from sucrose density gradients, activity was found predominantly at a density containing rough microsomes, with additional activities in the soluble and other fractions. Epoxidase activity was 2–4-times greater in the nitrogen-fixing nodules than in roots. This demonstration of active epoxidation indicates the capacity of nodules to detoxify other pesticides and xenobiotics.     

Occurrence and some properties of carbonic anhydrases from legume root nodules

Carbonic anhydrase activity (hydration of CO₂ was found in homogenates of leaves (116–500 units.mg^?1 protein) and root nodules (27–255 units.mg^?1 protein) from 8 legume genera inoculated in each case with a host specific Rhizobium. No enzyme, or only trace amounts (2–7 units.mg^?1 protein), were detected in root extracts, The enzymatic activity was inhibited in all cases by azide and acetazolamide. The sizes of nodule and leaf carbonic anhydrases, estimated by gel filtration of partially purified preparations from Phaseolus vulgaris, were around 45 000 and 205 000 respectively. These enzymes also differed in sensitivity to inhibitors. More than 99% of the activity present in Vicia faba nodules was recovered as a soluble enzyme and only a trace was located in the isolated bacteroids.     

添加葡萄糖和淀粉对盆栽甜樱桃根区土壤碳代谢及根功能的影响

在1年生盆栽甜樱桃土壤中添加葡萄糖和淀粉(4 g·kg^-1),以不添加外源碳为对照,处理后0~60 d内定期采根区土样测定土壤微生物生物量碳、蔗糖酶和淀粉酶活性以及微生物群落功能多样性,处理后第30天测定根系呼吸速率、呼吸途径和根系活力.结果表明： 添加葡萄糖后,土壤蔗糖酶活性及微生物生物量碳均表现为先升高再降低,峰值分别出现在处理后第15天及第7天,分别高于对照14.0%和13.1%,土壤有机质含量表现为先升高再降低再缓慢回升;添加淀粉后显著提高了土壤淀粉酶活性,第15天时为对照的8.5倍,土壤微生物生物量碳除在第7天低于对照外,其余时期均高于对照,土壤有机质含量表现为先升高再下降,处理后第60天高于对照19.8%.BIOLOG分析表明,处理后第15天平均吸光度（AWCD）值及微生物活性均达到最大值,表现为淀粉>葡萄糖>对照.处理后第30天,葡萄糖处理显著增加了土壤微生物对碳水化合物类、羧酸类、氨基酸类、酚酸类和胺类碳源的利用,淀粉处理显著增加了土壤微生物对碳水化合物类、羧酸类、聚合物类和酚酸类碳源的利用.处理后第30天,葡萄糖处理甜樱桃根系总呼吸速率分别较对照及淀粉处理提高21.4%和19.4%,根系活力分别提高65.5%和37.0%.添加葡萄糖和淀粉影响了甜樱桃根区土壤稳定碳源及不稳定碳源的代谢过程,整体上提高了土壤微生物活性,增强了甜樱桃根系呼吸速率及根系活力.     

Phosphorus sources and availability modify growth and distribution of root clusters and nodules of native Australian legumes

A variety of native Western Australian legumes produced root clusters in sand culture confirming field and published observations. In general, these legumes grew equally well when supplied with organic or inorganic sources of phosphorus. The nitrogen content of shoots and roots varied little among treatments for all species, however, phosphorus content was always greater in plants supplied with inositol‐P. The plasticity of root growth in response to localized placement of organic and inorganic sources of phosphorus was demonstrated using a simple ‘split root’ technique. Total root dry weight was, on average, more than doubled in P‐amended sand when compared with non‐amended sand. Root clusters tended to be produced in areas of relatively high phosphorus concentration and nodules in areas of low phosphorus concentration. Levels of phosphorus in lateral roots grown in P‐amended sand were significantly different from lateral roots grown in the corresponding non‐amended sand. Growth increases averaging 70% for white sand to over 100% for yellow sand indicated a large degree of ‘plasticity’ in roots under conditions of heterogeneous supply of phosphorus. Spatially exclusive development of organs for the acquisition of nutrients is discussed in relation to requirements for carbon in organ production and maintenance.     

Analysis of the subcellular localisation of lipoxygenase in legume and actinorhizal nodules

Plant lipoxygenases (LOXs; EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids, linoleic (18:2) and α-linolenic acid (18:3(n-3)) and are involved in processes such as stress responses and development. Depending on the regio-specificity of a LOX, the incorporation of molecular oxygen leads to formation of 9- or 13-fatty acid hydroperoxides, which are used by LOX itself as well as by members of at least six different enzyme families to form a series of biologically active molecules, collectively called oxylipins. The best characterised oxylipins are the jasmonates: jasmonic acid (JA) and its isoleucine conjugate that are signalling compounds in vegetative and propagative plant development. In several types of nitrogen-fixing root nodules, LOX expression and/or activity is induced during nodule development. Allene oxide cyclase (AOC), a committed enzyme of the JA biosynthetic pathway, has been shown to localise to plastids of nodules of one legume and two actinorhizal plants, Medicago truncatula, Datisca glomerata and Casuarina glauca, respectively. Using an antibody that recognises several types of LOX interspecifically, LOX protein levels were compared in roots and nodules of these plants, showing no significant differences and no obvious nodule-specific isoforms. A comparison of the cell-specific localisation of LOXs and AOC led to the conclusion that (i) only cytosolic LOXs were detected although it is generally assumed that the (13S)-hydroperoxy α-linolenic acid for JA biosynthesis is produced in the plastids, and (ii) in cells of the nodule vascular tissue that contain AOC, no LOX protein could be detected.     

Calcium-oxalate crystals and crystal cells in determinate root nodules of legumes

Early reports of the presence of calciumoxalate crystals in the cortices ofPhaseolus vulgaris root nodules have been confirmed. Crystals were found in all six genera examined (Cajanus, Desmodium, Glycine, Lespedeza, Phaseolus, Vigna) that have determinate nodules and export ureides. They were absent from six genera examined that have indeterminate nodules and export amides. The possible physiological significance of these structures is discussed.     

Molecular and serological comparisons of purified xanthine dehydrogenases from root nodules of three ureide-producing legume species

Xanthine dehydrogenase (XDH, EC 1.2.1.37) was immunopurified from root nodules of three legume species, soybean [ Glycine max (L.) Merr. cv. Pella], cowpea [ Vigna unguiculata (L.) Walp. cv. California Black Eye], and lima bean [ Phaseolus lunatus L. Henderson]. Polyclonal antibodies raised against each enzyme and monoclonal antibodies raised against soybean XDH were used to compare the three enzymes serologically. Double diffusion and enzyme-linked immunosorbent assays with polyclonal and monoclonal antibodies showed that the cowpea and lima bean enzymes are very similar immunologically but both differ measurably from soybean. Amino acid compositions of the legume nodule XDHs are presented as well. Although relatedness between these enzymes can be detected by immunological crossreactivity, each XDH has unique epitopes that can be used to distinguish the three proteins.     

Sterols of roots and nitrogen fixing root nodules of vicia faba

Sterols of both roots and nodules of Vicia faba were found to be mainly 24-methylcholesterol, 24-ethyl-5,22-cholestadien-3β-ol and 24-ethylcholesterol. Nodules contained nearly twice the quantity of sterols present in roots, mainly due to an increase in 24-ethylcholesterol from about 40–60% of the total sterol content of root extract to over 80% of nodule extracts. Insignificant amounts of sterol were detected in nodule bacteroid fractions and in free-living Rhizobium leguminosarum.     

Nitrate metabolism in roots and nodules of Vicia faba in response to exogenous nitrate

Activities of nitrate reduction enzymes, nitrate reductase activity (NRA) and nitrite reductase activity (NiRA) from roots and nodules of 5 mutant genotypes and one commercial cultivar (Alameda) of faba bean ( Vicia faba L. var. minor) grown in the presence of N₂ alone or with additional NO⁻₃ in the medium have been studied. A naturally occurring mutant (VFM109) with impaired ability to reduce nitrate in its nodules is described. All the other cultivars of V. faba showed nodule NRA, although the range was very wide, from almost negligible (VFM72) up to 2 μmol h⁻¹ (g FW)⁻¹. This activity was entirely of plant origin. Root NRA also ranged widely accross cultivars. However, the level of activity expressed as well as the response of NRA to nitrate followed a pattern opposite to that observed in nodules. Roots and nodules of all cultivars showed very high rates of NiRA, respectively 50 and 150-fold higher than NRA, thus precluding accumulation of nitrite in these tissues. Root enzymes were significantly stimulated by nitrate while negative (NRA) or little effect (NiRA) was found for nodules. Nitrate and nitrite reduction are carried out by inducible enzymes in roots of V. faba and by constitutive enzymes in nodules, indicating that there may be different forms of these enzymes in each tissue. Differences in the plant genotype were a major cause of the variability in nitrate and nitrite reduction by nodulated root systems of V. faba .     

Aerial root nodules in the tropical legume,Pentaclethra macroloba

Summary Symbiotic nitrogen fixation in angiosperms normally occurs in buried root nodules and is severely inhibited in flooded soils. A few plant species, however, respond to flooding by forming nodules on stems, or, in one case, submerged roots with aerenchyma. We report here the novel occurrence of aerial rhizobial nodules attached to adventitious roots of the legume,Pentaclethra macroloba, in a lowland tropical rainforest swamp in Costa Rica. Swamp sapdings (1–10 cm diameter) support an average 12 g nodules dry weight per plant on roots 2–300 cm above water, and nodules remain in aerial positions at least 6 months. Collections from four swamp plants maintained linear activity rates (3–14 moles C₂H₄/g nodule dry weight/hr) throughout incubations for 6 and 13 hrs; excised nodule activity in most legumes declines after 1–2 hrs. Preliminary study of the anatomy and physiology suggest aerial nodules possess unusual features associated with tolerance to swamp conditions. High host tree abundance and nodulation in the swamp compared to upland sites indicate the aerial root symbiosis may contribute more fixed nitrogen to the local ecosystem than the more typical buried root symbiosis.