Rates of activity
Alternative substrates for nitrogenase
Many compounds that contain triple bonds can be reduced by nitrogenase.
Acetylene is the most useful of these alternative substrates for the measurement of nitrogenase activity as ethylene is produced (Dilworth, 1966) and this can readily be quantified by gas chromatography. Whilst fixation of N by nitrogenase always results in the concomitant formation of hydrogen (Chapter 3), this does not occur during reduction of acetylene to ethylene:
NºN + 8H++ 8e-®2NH3+ H2
HCºCH + 2H++ 2e-®H2C = CH2
acetylene ethylene
The theoretical conversion ratio for acetylene (C2H2) reduced to N2fixed is thus 4 mol to 1 mol.
The use of acetylene for welding means that it is widely available, or can easily be made by reacting calcium carbide (CaC3) with water. The acetylene reduction assay of nitrogenase activity is carried out by incubating the test material in an atmosphere containing 10% acetylene in a closed vessel. The amount of ethylene (C2H4) produced after a period of incubation is then measured by gas chromatography. Fre- quently the nitrogenase activity, or acetylene reduction activity (ARA), is expressed directly as mM C2H4 produced per plant (or per nodule) h-1. Alternatively the amount of N2that would have been fixed (since reduction of acetylene always pre- cludes reduction of N2by nitrogenase) is calculated either by using the theoretical
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conversion ratio of 4 : 1 or by calculating the actual conversion ratio using a direct measurement of the rate of N2-fixation by15N2incorporation as described above, in parallel with an acetylene reduction assay.
Measurement of ARA is undoubtedly very useful as a rapid method for detection of nitrogenase activity and for measurement of enzyme rates in simple laboratory systems with free-living bacteria. As no N2 is actually being fixed by nitrogenase when acetylene is present, prolonged incubation periods can lead to N starvation in cultures, which in turn can cause further synthesis of nitrogenase and artificially high rates of N2-fixation (David and Fay, 1977). Much greater problems are associated with using ARA to quantify amounts of N2fixed and these problems warrant detailed description.
ARA measurements of ‘associative’ N2-fixation
Application of acetylene reduction assays for measurement of N2-fixation in soil is complicated by the effects of acetylene on other microbial processes. For example, acetylene blocks the last steps of denitrification (Balderstonet al., 1976) and autotrophic nitrification (Hynes and Knowles, 1978) so efficiently that it is used in experiments designed to measure these processes.
In addition, acetylene blocks bacterial oxidation of ethylene in soil so that
‘endogenous’ ethylene accumulates (de Bont, 1976; Nohrstedt, 1976). Thus control treatments used to estimate background concentrations of ethylene in soil, in which ethylene accumulation is measured in the absence of acetylene, greatly underestimate the accumulation of endogenous ethylene that occurs in the presence of acetylene.
Witty (1979) demonstrated this clearly by using14C-labelled acetylene for ARA mea- surements of N2-fixation in soil cores. Only half of the ethylene that accumulated over the incubation period was14C-labelled; the remaining unlabelled ethylene must have come from the soil.
Other methods for estimation of endogenous ethylene accumulation were sug- gested by Nohrstedt (1983). A small concentration (0.05%) of acetylene can be used in the control treatments; this is sufficient to block ethylene oxidation and thus produce the increase in ethylene that is not due to nitrogenase activity and yet is too low to divert a significant proportion of nitrogenase activity from N2-fixation to acetylene reduction. A further method is to include controls in which 10% acetylene is added together with 2% carbon monoxide. This inhibits nitrogenase activity whilst having little effect on other microbial processes, thus allowing N2-fixation and endogenous ethylene accumulation to be distinguished.
The14C-labelled acetylene method has been demonstrated to be more accurate than the carbon monoxide inhibition method, although the latter method is more suitable for routine use (Tann and Skujins, 1985).
Other problems in the application of acetylene reduction assays for measure- ment of low rates of nitrogenase activity in soil result from the differences in solubility and rates of diffusion of acetylene, ethylene and N2 (van Berkum and Bohlool, 1980). Given these problems it is suggested that the acetylene reduction assay should not be used for estimating N2-fixation with non-legumes.
ARA measurements of N2-fixation in legume nodules
The acetylene reduction assay has frequently been used to quantify N2-fixation in field- or glasshouse-grown legumes, but there is considerable evidence which suggests that few of these measurements bear much relation to reality (Witty and Minchin, 1988). The most common method of measuring ARA in legume nodules is to uproot the plants, separate the shoot and roots and then incubate the roots plus attached nodules in a closed container under 10% acetylene. When this ‘static’ assay of ARA was developed, apparently linear rates of ethylene production were measured over incubation periods of 30 min to 1 h.
The development of more sophisticated flow-through incubation systems, where the nodules are held in a constantly flowing gas stream into which acetylene can be introduced (Wittyet al., 1983), has allowed more detailed investigation of the time course of ARA. On introduction of acetylene the rate of ethylene production rises quickly to a maximum value and then begins to decline after a few minutes, reaching a lower steady rate after 30 min (Fig. 4.1a). This ‘acetylene-induced decline’
in nitrogenase activity is accompanied by a similar decline in nodule respiration and is thought to be brought about by changes in the diffusion resistance of the nodule to oxygen (Chapter 3). Ethylene production measured in this way can be plotted as the total cumulative ethylene produced over the period of the assay just as in the ‘static’
assay (Fig. 4.1b), but the more detailed analysis reveals that the true rate (i.e. the initial rate) of nitrogenase activity is much greater than that measured in the static assay in an enclosed incubation vessel.
There are several additional sources of error in ARA measurements of nitro- genase activity. One is the simple difficulty of ensuring that all nodules are recovered from the soil, particularly in field-grown plants. A second is the fact that ARA measurement reflects nitrogenase activity only at a particular point in time – over the duration of the assay – which is far from being a measurement of the amount of N2
Fig. 4.1. The acetylene reduction assay in a flow-through system. (a) The change in rate of ethylene production (mM C2H4per plant min-1) showing the acetylene- induced decline. (b) The cumulative ethylene production (mM C2H4per plant).
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fixed over an entire growing season. Given the large diurnal fluctuations, and the wide variations in rates of nitrogenase activity (as assessed by ARA) commonly found between harvest intervals, calculation of the total N2fixed from spot measurements of ARA (or other methods that give an instantaneous rate of N2-fixation) is bound to be inaccurate unless estimated very frequently. Moreover, as the acetylene reduction assay on detached roots is a destructive assay, the same plants can never be assayed twice. A third problem is that it has now been shown that virtually any disturbance, from one as mild as shaking soil off the roots to one as dramatic as removing the shoots, can cause a marked reduction in the rate of nitrogenase activity (Minchin et al., 1986). Finally, the theoretical conversion factor of 4 : 1 mol C2H4reduced to N2fixed is often assumed to be valid, and is used directly in calculations. However, experimental measurements of this ratio show that the true conversion factor can vary widely and is often between 5 : 1 and 7.5 : 1 (Witty and Minchin, 1988).
It can be tempting to assume that such errors will be uniform across different experimental treatments and thus do not affect the validity of purely comparative measurements. Unfortunately this is not the case. The magnitude of each of these errors can be influenced by treatments that are commonly compared using ARA measurements (Table 4.2), including the effect of different rhizobial strains or legume genotypes. In the light of these errors, use of the ‘static’ acetylene reduction assay can be justified only for simple demonstration that nodules are fixing N2and never for accurate measurement or even just relative comparison of nitrogenase activity between treatments.
For physiological studies of N2-fixation, flow-through incubation chambers can be used to measure and compare rates of nitrogenase activity by acetylene reduction.
This can be done on detached root systems, or on whole plants if the plants are grown in special pots that can be sealed into a flow-through gas system. Such experiments on detopped root systems have yielded useful information (e.g. Wittyet al., 1983) but measurements should be made on undisturbed plants if true in situ rates of N2-fixation are to be observed (Minchinet al., 1986). Flow-through ARA chambers have been developed for use in the field (Denisonet al., 1983; Sheehyet al., 1991) but problems of restricted root growth and other technical difficulties prevent their widespread use.
Vessey (1994) argued that the static ARA assay still has valuable applications, despite all of the problems listed above, for measuring relative differences between nodulated legumes in the field. Others disagree (e.g. Minchinet al., 1994) and sug- gest that there are few circumstances where ARA has benefits over simple measure- ments of yield or N accumulation. It is important to remember that much of the knowledge on N2-fixation accumulated before the 1980s was obtained using the static acetylene reduction assay. Given present understanding of the limitations of this method, some caution in accepting conclusions from earlier research is certainly justified.
Analysis of N-transport compounds
Newly fixed N is assimilated in the legume nodule and then transported to other parts of the plant either as amides or as ureides (Chapter 3). Nitrogen is also absorbed
from the soil, predominantly as nitrate but also as ammonium. Nitrate is either reduced to ammonium in the roots (by the sequential action of the enzymes nitrate reductase and nitrite reductase) and assimilated into amino acids before being transported, or is transported directly as nitrate to the stem before reduction and assimilation. Ammonium ions, on the other hand, are always rapidly assimilated before being transported, as they are toxic. Ureide production is restricted to certain taxonomic groups of legumes (Table 4.3). Of the papilionoid legumes, all members of the tribesPhaseoleae(except perhapsErythrinaspp. which has a somewhat anoma- lous taxonomic position) andDesmodieaeexamined so far transport the products of N2-fixation as ureides, whilst all members of the Aeschynomeneae, Robinieae, TrifolieaeandVicieaetransport amides.1(Footnote on p. 81.) Among theCaesalpini- oideaeandMimosoideaethere are as yet no confirmed reports of species that transport the products of N2-fixation as ureides. Although the xylem sap of actinorhizal plants Error in measurement
of absolute rates Causes of variation Treatments responsible Decrease in activity
caused by disturbance and by the acetylene- induced decline in nitrogenase activity
Lack of calibration of C2H2: N2conversion ratios
Incomplete recovery of nodules
Alteration of the oxygen diffusion resistance of the nodule
In comparative assays, differences in the initial diffusion resistance can alter the extent of the error
C2H2: N2ratios greater than 4 : 1
Failure to excavate all of root system
General:
any plant disturbance washing of roots Treatment dependent:
defoliation water stress temperature
plant species or genotype rhizobial strain
Treatment-dependent differences in electron allocation between N2and H2due to:
temperature irradiance
plant species or genotype rhizobial strain
uptake hydrogenase Treatment-dependent differences in nodule distribution and soil structure as a result of:
plant spacing or age fertilizer applications plant species or genotype rhizobial strain
Table 4.2. Sources of error in acetylene reduction assays of N2-fixation. (After Witty and Minchin, 1988.)
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Type of
legumes Ureide producers Amide producers
Grain legumes
Forage legumes
Shrub and tree legumes
Phaseoleae:
Cajanus cajan,Glycine max, Lablab purpureus,
Macrotyloma geocarpum, Phaseolus lunatus,P. vulgaris, Psophocarpus tetragonolobus, Vigna aconitifolia,
V. angularis,V. mungo, V. radiata,V. subterranea, V. trilobata,V. umbellata, V. unguiculata
Indigofereae:
Cyamopsis tetragonoloba Desmodieae:
Desmodium discolor, D. uncinatum Phaseoleae:
Calopogonium caeruleum, Centrosema pubescens, Macroptilium atropurpureum, Macrotyloma uniflorum, Pueraria javanica, P. phaseoloides Desmodieae:
Codariocalyx gyroides, Desmodium rensonii
Aeschynomeneae:
Arachis hypogaea Cicereae:
Cicer arietinum Genisteae:
Lupinus mutabilis Vicieae:
Lathyrus sativus,Lens culinaris, L. esculenta,Vicia faba
Aeschynomeneae:
Arachis glabrata, A. pintoi,Zorniaspp.
Crotalarieae:
Crotalariaspp.
Trifolieae:
Trifolium pratense, T. repens,T. subterraneum
Aeschynomeneae:
Aeschynomene indica Acacieae:
Acacia alata, A. auriculiformis, A. extensa,A. pulchella, A. unisauvis
Ingeae:
Calliandra calothyrsus Mimoseae:
Leucaena diversifolia, L. leucocephala, L. macrophylla, Prosopis juliflora Robinieae:
Gliricidia sepium, Sesbania grandiflora, S. sesban,S. rostrata Table 4.3. Tropical legumes that transport the products of N2-fixation mainly as ureides or amides. (After Ledgard and Peoples, 1988; Peopleset al., 1989a, 1991a;
Peoples and Herridge, 1990; Yoneyama and Kondo, 1990; Herridgeet al., 1996.)
such asAlnusandCasuarinahave been shown to contain the ureide citrulline, this appears not to be related specifically to N2-fixation (Walshet al., 1984).
Ureide transporters tend to have a low activity of nitrate reductase in the roots and so the majority of the nitrate absorbed is transported directly to the shoots. The majority of the N in the xylem sap of a ureide-producing legume that is fixing most of its N will therefore be in the form of ureides, whilst if the same species is absorbing most of its N from the soil, the majority of N in the sap will be in the form of nitrate or, to a lesser extent, amides. The proportion of N in the xylem sap that is present as ureides has therefore been developed as an index of N2-fixation (McClureet al., 1980; Pateet al., 1980).
To carry out ureide assays, xylem sap is obtained by decapitating plants and collecting the bleeding sap from the cut stump. The contents of ureides, total a-amino N and nitrate in the sap are estimated by colorimetric assays and the results are expressed as the relative % ureide content (Peopleset al., 1989a). To obtain actual measurements of N2-fixation, the relative ureide content must be calibrated against the % N2fixed by using another method for the measurement of N2-fixation. This is usually done by growing the test legumes in pots in the glasshouse, feeding them with increasing nitrate concentrations to progressively suppress nodulation and N2-fixation (Chapter 3), and measuring the ureide content of plants that are dependent on N2-fixation to different degrees (Fig. 4.2a). This assay does not measure the total N2fixed – it only provides an estimate of the proportion of plant N derived from N2-fixation. Sequential measurements of N accumulation together with repeated estimates of xylem sap composition are required to estimate total amounts of fixed N (Herridgeet al., 1990).
Limitations of the technique include the variation in composition of the sap with the age of the plants and the difficulty in extraction of sap (Peoples et al., 1989b). Bleeding sap can be collected from cut stumps of plants only if there is sufficient available soil moisture. Sap can also be extracted from segments of shoot tissue by application of a mild vacuum or, alternatively, soluble N compounds can be extracted in aqueous solution, although the composition of such extracts may differ from that of the bleeding sap and may require separate calibration (Herridge and Peoples, 1990). The method does not appear to be valid once seed filling begins due to remobilization of N within the plant (Avelineet al., 1995). Nevertheless, the method has been shown to give estimates of N2-fixation that correlate very well with estimates obtained using other methods (Fig. 4.2b). A simpler approach is to collect leaf or preferably petiole samples and extract the ureides with water for analysis (Peopleset al., 1989a; Alveset al., 2000).
Whilst the above method can only be used with legumes that transport the products of N2-fixation as ureides, the relative concentrations of nitrate and amide-N
1 Ureides have been detected as minor components of the xylem sap in several species (e.g.
Arachis hypogaea, Erythrina variegata, Flemingia macrophylla, Gliricidia sepium, Paraseri- anthes falcataria, Sesbaniaspp., Stylosanthes hamata) where they are not related to N2- fixation. In addition unrelated coloured products form when the sap of some species are assayed for ureides which has probably led to incorrect reports of some species as ureide producers (Peopleset al.,1991a).
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were also shown to be useful indicators of dependence on N2-fixation in chickpea, groundnut, lentils and pea (Peoples et al., 1986, 1987). Clearly this depends on nitrate absorbed from the soil not being assimilated in the roots, and this relationship was found not to hold in faba bean (Vicia faba), which had a large proportion of its nitrate reductase activity in the roots.
Although there are obvious limitations to the widespread use of the analysis of N-transport compounds for estimating N2-fixation, there may be situations in which the method has particular advantages. If a method could be developed for tapping the xylem sap of tree legumes so that sap could be sampled frequently without killing the tree, then this could provide a way of assessing seasonal contributions from N2-fixation in the field. Unfortunately, of the legume trees that have been investi- gated, few of those widely used in agroforestry transport the products of N2-fixation as ureides (Table 4.3 and Chapter 11).
Fig. 4.2. (a) Glasshouse calibra- tion of the ureide method for measuring N2-fixation in soybean against15N isotope dilution measurements for soybean (Herridge and Peoples, 1990).
(b) Comparison of field measure- ments of N2-fixation in soybean made using either the ureide method or the15N natural abundance method. The different symbols represent measurements from different fields within the same experimental station.
(From Herridgeet al., 1990.)
Integrated measurements
The15N isotope dilution method
One of the most widely used methods for integrated measurements of N2-fixation is based on the principle of15N isotope dilution. An understanding of the terminology involved in the description of stable isotope use is necessary background for an explanation of this method and a number of definitions are presented in Table 4.4.
The content of the N present in a substance that consists of the stable isotope
15N is normally expressed as the proportion of15N atoms present (i.e. atom %15N).
Nitrogen in the atmosphere is virtually all 14N2 and the natural 15N content or natural abundance of the atmosphere has been shown to be a constant 0.3663 atom
%15N throughout the world (Mariotti, 1983). The remaining 99.6336% are there- fore14N atoms. Any substance that has an atom %15N greater than that of the atmo- sphere is said to be enriched with15N and the15N-enrichment is expressed as the atom %15N above that of the atmosphere, or atom %15N excess. Similarly a material depleted in15N has an atom %15N below that of the atmosphere. When discussing the15N enrichment of natural materials, that is their natural abundance which is usually a small value of atom %15N excess, then the termd15N is used (Table 4.4).
If a plant is grown in conditions where its sole source of N is fertilizer N that is entirely composed of15N (100 atom %15N), then all of the N in the plant (apart from the amount that is originally present in the seed or bacterial inoculum) will be
15N. If the plant is able to fix dinitrogen (14N2) from the atmosphere, then the plant will have an atom %15N that is less than that of the fertilizer (i.e. less than 100 atom %15N). This difference can be used to calculate the proportion of N derived from N2-fixation and is the underlying principle of the isotope dilution method for measurement of N2-fixation. The exact calculation of the amount of N from N2-fixation is shown in equation 2a in Table 4.5. For example, if the N2-fixing plant has a final15N-enrichment of 75 atom %15N, then:
Term Definition
Atom %15N Natural abundance Natural abundance of the atmosphere
Atom %15N excess
15N-enriched nitrogen
15N-depleted nitrogen
d15N (in parts per thousand, or ‰)
Abundance of15N atoms as a percentage of the total = (15N/(15N +14N))´100%
Atom %15N present in natural materials Atom %15N of the atmosphere = 0.3663 atom
%15N
The percentage of15N above that in the atmosphere = atom %15N-0.3663 Nitrogen with an atom %15N above that of the atmosphere
Nitrogen with an atom %15N below that of the atmosphere
((Rsample-Rreference)/Rreference)´1000, where R = ((15N +14N)/(14N +14N))
Table 4.4. Terms used in the description of15N stable isotope methods.
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