Continued part 1, part 2 of ebook Plant biochemistry (Third edition) provide readers with content about: nitrogen fixation enables the nitrogen in the air to be used for plant growth; sulfate assimilation enables the synthesis of sulfur-containing substances; phloem transport distributes photoassimilates to the various sites of consumption and storage;...
11 Nitrogen fixation enables the nitrogen in the air to be used for plant growth In a closed ecological system, the nitrate required for plant growth is derived from the degradation of the biomass In contrast to other plant nutrients (e.g., phosphate or sulfate), nitrate cannot be delivered by the weathering of rocks Smaller amounts of nitrate are generated by lightning and carried into the soil by rain water (in temperate areas about kg N/ha per year) Due to the effects of civilization (e.g., car traffic, mass animal production, etc.), the amount of nitrate, other nitrous oxides and ammonia carried into the soil by rain can be in the range of 15 to 70 kg N/ha per year Fertilizers are essential for agricultural production to compensate for the nitrogen that is lost by the withdrawal of harvest products For the cultivation of maize, for instance, per year about 200 kg N/ha have to be added as fertilizers in the form of nitrate or ammonia Ammonia, the primary product for the synthesis of nitrate fertilizer, is produced from nitrogen and hydrogen by the Haber-Bosch process: 3H + N Æ 2NH ( DH - 92,6kJ/mol) Because of the high bond energy of the N∫N triple bond, this synthesis requires a high activation energy and is therefore, despite a catalyzator, carried out at a pressure of several hundred atmospheres and temperatures of 400°C to 500°C Therefore it involves very high energy costs The synthesis of nitrogen fertilizer amounts to about one-third of the total energy expenditure for the cultivation of maize If it were not for the production of nitrogen fertilizer by Haber-Bosch synthesis, large parts of the world’s population could no longer be fed Using “organic cycle” agriculture, one 309 310 11 Nitrogen fixation enables the nitrogen in the air to be used for plant growth hectare of land can feed about 10 people, whereas with the use of nitrogen fertilizer the amount is increased fourfold The majority of cyanobacteria and some bacteria are able to synthesize ammonia from nitrogen in air A number of plants live in symbiosis with N2-fixing bacteria, which supply the plant with organic nitrogen In return, the plants provide these bacteria with metabolites for their nutrition The symbiosis of legumes with nodule-inducing bacteria (rhizobia) is widespread and important for agriculture Legumes, which include soybean, lentil, pea, clover, and lupines, form a large family (Leguminosae) with about 20,000 species A very large part of the legumes have been shown to form a symbiosis with rhizobia In temperate climates, the cultivation of legumes can lead to an N2 fixation of 100 to 400 kg N2/ha per year Therefore legumes are important as green manure; in crop rotation they are an inexpensive alternative to artificial fertilizers The symbiosis of the water fern Azolla with the cyanobacterium Nostoc supplies rice fields with nitrogen N2-fixing actinomycetes of the genus Frankia form a symbiosis with woody plants such as the alder or the Australian casuarina The latter is a pioneer plant on nitrogen-deficient soils 11.1 Legumes form a symbiosis with nodule-inducing bacteria Initially it was thought that the nodules of legumes (Fig 11.1) were caused by a plant disease, until their function in N2 fixation was recognized by H Hellriegel and H Wilfarth in 1888 They found that beans containing these nodules were able to grow without nitrogen fertilizer The nodule-inducing bacteria include, among other genera, the genera Rhizobium, Bradyrhizobium, and Azorhizobium and are collectively called rhizobia The rhizobia are strictly aerobic gram-negative rods, which live in the soil and grow heterotrophically in the presence of organic compounds Some species (Bradyrhizobium) are also able to grow autotrophically in the presence of H2, although at a low growth rate The uptake of rhizobia into the host plant is a controlled infection The molecular basis of specificity and recognition is still only partially known The rhizobia form species-specific nodulation factors (Nod factors) These are lipochito-oligosaccharides that acquire a high structural specificity (e.g., by acylation, acetylation, and sulfatation) They are like a security key with many notches and open the house of the specific host with which the rhizobia associate The Nod factors bind to specific receptor kinases of the host, 11.1 Legumes form a symbiosis with nodule-inducing bacteria 311 Firgure 11.1 Root system of Phaseolus vulgaris (bean) with a dense formation of nodules after infection with Rhizobium etli (By P Vinuesa-Fleischmann and D Werner, Marburg.) which are part of signal transduction chains (section 19.1) In this way the “key” induces the root hair of the host to curl and the root cortex cells to divide, forming the nodule primordium After the root hair has been invaded by the rhizobia, an infection thread forms (Fig 11.2), which extends into the cortex of the roots, forms branches there, and infects the cells of the nodule primordium A nodule thus develops from the infection thread The morphogenesis of the nodule is of similarly high complexity to that of any other plant organ such as the root or shoot The nodules are connected with the root via vascular tissues, which supply them with substrates formed by photosynthesis The bacteria, which have been incorporated into the plant cell, are enclosed by a peribacteroid membrane (also called a symbiosome membrane), which is formed by the plant The incorporated bacteria are thus separated from the cytoplasm of the host cell in a so-called symbiosome (Fig 11.3) In the symbiosome, the rhizobia differentiate to bacteroids The volume of these bacteroids can be 10 times the volume of individual bacteria Several of these bacteroids are surrounded by a peribacteroid membrane 312 Figure 11.2 Controlled infection of a host cell by rhizobia is induced by an interaction with the root hairs The rhizobia induce the formation of an infection thread, which is formed by invagination of the root hair cell wall and protrudes into the cells of the root cortex In this way the rhizobia invaginate the host cell where they are separated by a peribacteroid membrane from the cytosol of the host cells The rhizobia grow and differentiate into large bacteroids 11 Nitrogen fixation enables the nitrogen in the air to be used for plant growth Peribacteroid membrane Rhizobia Root hair cell Infection thread (invagination of root hair membrane) Infected cell Figure 11.3 Electron microscopic cross section through a nodule of Glycine max cv Caloria (soybean) infected with Bradyrhizobium japonicum The upper large infected cell shows intact symbiosomes (S) with one or two bacteroids per symbiosome In the lower section, three noninfected cells with nucleus (N), central vacuole (V), amyloplasts (A), and peroxisomes (P) are to be seen (By E Mörschel and D Werner, Marburg.) Rhizobia possess a respiratory chain with a basic structure corresponding to that of the mitochondrial respiratory chain (see Fig 5.15) In a Bradyrhizobium species, an additional electron transport path is formed during differentiation of the rhizobia to bacteroids This path branches at 11.1 Legumes form a symbiosis with nodule-inducing bacteria the cyt-bc1 complex of the respiratory chain and conducts electrons to another terminal oxidase, enabling an increased respiratory rate It is encoded for by symbiosis-specific genes The formation of nodules is due to a regulated interplay of the expression of specific bacteria and plant genes Rhizobia capable of entering a symbiosis contain a large number of genes, which are switched off in the free-living bacteria and are activated only after an interaction with the host, to contribute to the formation of an N2-fixing nodule The bacterial genes for proteins required for N2 fixation are named nif and fix genes, and those that induce the formation of the nodules are called nod genes The host plant signals its readiness to form nodules by excreting several flavonoids (section 18.5) as signal compounds These flavonoids bind to a bacterial protein, which is encoded by a constitutive (which means expressed at all times) nod gene The protein, to which the flavonoid is bound, activates the transcription of the other nod genes The proteins encoded by these nod genes are involved in the synthesis of the Nod factors mentioned previously Four so-called “general” nod genes are present in all rhizobia In addition, more than 20 other nod genes are known, which are responsible for the host’s specificity Those proteins required especially for the formation of nodules, and which are synthesized by the host plant in the course of nodule formation, are called nodulins These nodulins include leghemoglobin (section 11.2), the enzymes of carbohydrate degradation [including sucrose synthase (section 9.2)], enzymes of the citrate cycle and the synthesis of glutamine and asparagine, and, if applicable, also of ureide synthesis They also include an aquaporin of the peribacteroid membrane The plant genes encoding these proteins are called nodulin genes One differentiates between “early” and “late” nodulins Early nodulins are involved in the process of infection and formation of nodules, where the expression of the corresponding genes is induced in part by signal substances released from the rhizobia “Late” nodulins are synthesized only after the formation of the nodules In many cases, nodulins are isoforms of proteins found in other plant tissues Metabolic products are exchanged between bacteroids and host cells The main substrate provided by the host cells to the bacteroids is malate (Fig 11.4), formed from sucrose, which is delivered by the sieve tubes The 313 314 Figure 11.4 Metabolism of infected cells in a root nodule Glutamine and asparagine are formed as the main products of N2 fixation (see also Fig 11.5) 11 Nitrogen fixation enables the nitrogen in the air to be used for plant growth Sucrose HOST CELL Malate BACTEROID N2 Fixation N2 NH 4+ Glutamine Asparagine sucrose is metabolized by sucrose synthase (Fig 13.5), degraded by glycolysis to phosphoenolpyruvate, which is carboxylated to oxaloacetate (see Fig 10.11), and the latter is reduced to malate Nodule cells contain high activities of phosphoenolpyruvate carboxylase NH4+ is delivered as a product of N2 fixation to the host cell, where it is subsequently converted mainly into glutamine (Fig 7.9) and asparagine (Fig 10.14) and then transported via the xylem vessels to the other parts of the plant It was recently shown that alanine also can be exported from bacteroids The nodules of some plants (e.g., those of soybean) export the fixed nitrogen as ureides (urea degradation products), especially allantoin and allantoic acid (Fig 11.5) These compounds have a particularly high nitrogen to carbon ratio The formation of ureides in the host cells requires a complicated synthetic pathway First, inosine monophosphate is synthesized via the pathway of purine synthesis, which is present in all cells for the synthesis of AMP and GMP, and then it is degraded via xanthine and ureic acid to the ureides mentioned previously Malate taken up into the bacteroids is oxidized by the citrate cycle (Fig 5.3) The reducing equivalents thus generated are the fuel for the fixation of N2 11.1 Legumes form a symbiosis with nodule-inducing bacteria 315 Phosphoribosylpyrophosphate Glutamine Aspartate Glycine ATP Purinbiosynthesis O HN HC C N H2O + O2 O C N HN CH C N C O Ribose-P Inosine monophosphate C N H C N H2O2 O HN CH C N H C O Xanthine oxidase Xanthine C H C N N H C N H C O Uric acid 1/ O2 + H2O Uricase CO2 H2O NH2 C O H2N COO NH2 C C N N O H H H C Allantoinase Allantoate Figure 11.5 In some legumes (e.g., soy bean and cow pea), allantoin and allantoic acid are formed as products of N2 fixation and are delivered via the roots to the xylem Their formation proceeds first via inosine monophosphate by the purine synthesis pathway Inosine monophosphate is oxidized to xanthine and then further to ureic acid Allantoin and allantoic acid are formed by hydrolysis and opening of the ring Nitrogenase reductase delivers electrons for the nitrogenase reaction Nitrogen fixation is catalyzed by the nitrogenase complex, a highly complex system with nitrogenase reductase and nitrogenase as the main components (Fig 11.6) This complex is highly conserved and is present in the cytoplasm of the bacteroids From NADH formed in the citrate cycle, electrons are transferred via soluble ferredoxin to nitrogenase reductase The latter is a one-electron carrier, consisting of two identical subunits, which together form a 4Fe-4S cluster (see Fig 3.26) and contain two binding sites for ATP After reduction of nitrogenase reductase, two molecules of ATP bind to it, resulting in a conformational change of the protein, by which the redox potential of the 4Fe-4S cluster is raised from -0.25 to -0.40 V Following the transfer of an electron to nitrogenase, the two ATP molecules bound to O O H C N C N H N H H Allantoin C O 316 11 Nitrogen fixation enables the nitrogen in the air to be used for plant growth ATP 1e 1/ NADH Fdox Fe-4 S red E0' -0,25 V Nitrogenase complex Fe-4 S red ATP Dinitrogenase reductase 1/ NAD + + 1/2 H + Fdred Fe-4 S ox N2 E0' -0,40 V Fe-4 S ox ATP 1e 8e P Cluster Fe-Mo 2x Cofactor Fe-4 S Mo-7 Fe- S Dinitrogenase + H+ NH3 + H2 ADP + P Figure 11.6 The nitrogenase complex consists of the nitrogenase reductase and the nitrogenase Their structure and function are described in the text The reduction of one molecule of N2 is accompanied by the reduction of at least two protons to form molecular hydrogen the protein are hydrolyzed to ADP and phosphate, and then released from the protein As a result, the conformation with the lower redox potential is restored and the enzyme is again ready to take up one electron from ferredoxin Thus with the consumption of two molecules of ATP, one electron is transferred from NADH to nitrogenase by nitrogenase reductase N2 as well as H+ are reduced by nitrogenase Nitrogenase is an a2b2 tetramer The a and b subunits have a similar size and are similarly folded The tetramer contains two catalytic centers, probably reacting independently of each other, and each contains a so-called P cluster, consisting of two 4Fe-4S clusters and an iron molybdenum cofactor (FeMoCo) FeMoCo is a large redox center made up of Fe4S3 and Fe3MoS3, which are linked to each other via three inorganic sulfide bridges (Fig 11.7) A further constituent of the cofactor is homocitrate, which is linked via oxygen atoms of the hydroxyl and carboxyl group to molybdenum Another ligand of molybdenum is the imidazole ring of a histidine residue of the protein The function of the Mo atom is still unclear Alternative nitrogenases are known, in which molybdenum is replaced by vanadium or iron, but these nitrogenases are much more unstable than the nitrogenase containing FeMoCo The Mo atom possibly causes a more favorable geometry 11.1 Legumes form a symbiosis with nodule-inducing bacteria His NH S N O C O Mo OOC CH2 C O Homocitrate CH2 CH2 Fe S Fe S Fe S N N S Fe H S Fe S Fe Fe S S Fe3MoS3 Fe4S3 COO Three inorganic S-bridges and electron structure of the center It is not yet known how nitrogen reacts with the iron-molybdenum cofactor One possibility would be that the N2 molecule is bound in the cavity of the FeMoCo center (Fig 11.7) and that the electrons required for N2 fixation are transferred by the P cluster to the FeMoCo center Nitrogenase is able to reduce other substrates beside N2 (e.g., protons, which are reduced to molecular hydrogen): 2H + + 2e - ỉNitrogenase ỉỉỉỈ H During N2 fixation at least one molecule of hydrogen is formed per N2 reduced: 8H + + 8e - + N ỉNitrogenase ỉỉỉỈ 2NH + H Thus the balance of N2 fixation is at least: N + 4NADH + 4H + + 16ATP ỉ ỉỉỉỈ 2NH + H + 4NAD + + 16ADP + 16P In the presence of sufficient concentrations of acetylene, only this is reduced and ethylene is formed: HC ∫ CH + 2e - + 2H + ỉNitrogenase ỉỉỉỈ H C = CH This reaction is used to measure the activity of nitrogenase Why H2 evolves during N2 fixation is not known It may be part of the catalytic mechanism or a side reaction or a reaction to protect the active center against the 317 Figure 11.7 The ironmolybdenum cofactor consists of the fragments Fe4S3 and MoFe3S3, which are linked to each other by three inorganic sulfide bridges In addition, the molybdenum is ligated with homocitrate and the histidine side group of the protein The cofactor binds one N2 molecule and reduces it to two molecules of NH3 by successive uptake of electrons The position where N2 is bound in the cofactor has not yet been experimentally proven (After Karlin 1993.) 318 11 Nitrogen fixation enables the nitrogen in the air to be used for plant growth inhibitory effect of oxygen The formation of molecular hydrogen during N2 fixation can be observed in a clover field Many bacteroids, however, possess hydrogenases by which H2 is reoxidized by electron transport: 2H + O ỉHydrogenase ỉỉỉ ỉỈ H O It is questionable, however, whether this reaction is coupled in the bacteroids to the generation of ATP 11.2 N2 fixation can proceed only at very low oxygen concentrations Nitrogenase is extremely sensitive to oxygen Therefore N2 fixation can proceed only at very low oxygen concentrations The nodules form an anaerobic compartment Since N2 fixation depends on the uptake of nitrogen from the air, the question arises how is the enzyme protected against the oxygen present in air? The answer is that oxygen, which has diffused together with nitrogen into the nodules, is consumed by the respiratory chain contained in the bacteroid membrane Due to a very high affinity of the bacteroid cytochrome-a/a3 complex, respiration is still possible with an oxygen concentration of only 10-9 mol/L As described previously, at least a total of 16 molecules of ATP are required for the fixation of one molecule of N2 Upon oxidation of one molecule of NADH, about 2.5 molecules of ATP are generated by the mitochondrial respiratory chain (section 5.6) In the bacterial respiratory chain, which normally has a lower degree of coupling than that of mitochondria, only about two molecules of ATP may be formed per molecule of NADH oxidized Thus about four molecules of O2 have to be consumed for the formation of 16 molecules of ATP (Fig 11.8) If the bacteroids possess a hydrogenase, due to the oxidation of H2 formed during N2 fixation, oxygen consumption is further increased by half an O2 molecule Thus during N2 fixation, for each N2 molecule at least four O2 molecules are consumed by bacterial respiration (O2/N2 ≥ 4) In contrast, the O2/N2 ratio in air is about 0.25 This comparison shows that air required for N2 fixation contains in relation to nitrogen far too little oxygen The outer layer of the nodules is a considerable diffusion barrier for the entry of air The diffusive resistance is so high that bacteroid respiration is limited by the uptake of oxygen This leads to the astonishing situation that N2 fixation is limited by the influx of O2 for formation of the required ATP Experiments by the Australian Fraser Bergersen have presented evidence for ... Inosine monophosphate C N H C N H2O2 O HN CH C N H C O Xanthine oxidase Xanthine C H C N N H C N H C O Uric acid 1/ O2 + H2O Uricase CO2 H2O NH2 C O H2N COO NH2 C C N N O H H H C Allantoinase... half an O2 molecule Thus during N2 fixation, for each N2 molecule at least four O2 molecules are consumed by bacterial respiration (O2/N2 ≥ 4) In contrast, the O2/N2 ratio in air is about 0 .25 This... NADH + FADH2 NADH NADH O2 O2 20 % N2 N2 80% Ferredoxinred 16 ATP Respiratory chain H 2O Nitrogenase NH3 + H2 Peribacteroid membrane Plasma membrane of host cell Wall of nodule Figure 11.8 N2 fixation