Evaluation of N2-fixation and nitrogen economy of a maize/cowpea intercrop system using15N dilution methods

Yields of above ground biomass and total N were determined in summer-grown maize and cowpea as sole crops or intercrops, with or without supplementary N fertilizer (25 kg N ha⁻¹, urea) at an irrigated site in Waroona, Western Australia over the period 1982–1985. Good agreement was obtained between estimates of N₂ fixation of sole or intercrop cowpea (1984/85 season) based on the¹⁵N natural abundance and¹⁵N fertilizer dilution techniques, both in the field and in a glasshouse pot study. Field-grown cowpea was estimated to have received 53–69% of its N supply from N₂-fixation, with N₂-fixation onlyslightly affected by intercropping or N fertilizer application. Proportional reliance on N₂-fixation of cowpea in glasshouse culture was lower (36–66%) than in the field study and more affected by applied N. Budgets for N were drawn up for the field intercrops, based on above-ground seed yields, return of crop residues, inputs of fixed N and fertilizer N. No account was taken of possible losses of N through volatilization, denitrification and leaching or gains of N in the soil from root biomass. N₂-fixation was estimated tobe 59 kg N ha⁻¹ in the plots receiving no fertilizer N, and 73 kg N ha⁻¹ in plots receiving 25 kg N ha⁻¹ as urea. Comparable fixation by sole cowpea was higher (87 and 82 kg N ha⁻¹ respectively) but this advantage was outweighed by greater land use efficiency by the intercrop than sole crops.     

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Identification of essential sulfhydryl residues in the primary sequence of the enzyme

The kinase and sugar phosphate exchange reactions of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by treatment with 5'-p-fluorosulfonylbenzoyladenosine or 8-azido-ATP, but activity could be restored by the addition of dithiothreitol. This inactivation was accompanied by incorporation of 5'-p-sulfonylbenzoyl[8-14C]adenosine into the enzyme that was not released by the addition of dithiothreitol. The lack of effect of ATP analogs on the ATP/ADP exchange or on bisphosphatase activity and reversal of their effects on the kinase and sugar phosphate reactions by dithiothreitol suggest that 1) they reacted with sulfhydryl groups important for sugar phosphate binding in the kinase reaction, and 2) the inactivation of the kinase by these analogs involves a specific reaction that is not related to their general mechanism of attacking nucleotide-binding sites. In addition, alkylation of the enzymes' sulfhydryls with iodoacetamide prevented inactivation by 5'-p-fluorosulfonylbenzoyladenosine, suggesting that the same thiols were involved. o-Iodosobenzoate inactivated the kinase and sugar phosphate exchange; the inactivation was reversed by dithiothreitol; but there was no effect on the bisphosphatase or nucleotide exchange, indicating that oxidation occurred at the same sulfhydryl that are associated with sugar phosphate binding. ATP or ADP, but not fructose 6-phosphate, protected these groups from modification by 5'-p-fluorosulfonylbenzoyladenosine, 8-azido-ATP, and o-iodosobenzoate. ATP also induced dramatic changes in the circular dichroism spectrum of the enzyme, suggesting that adenine nucleotide protection of thiol groups resulted from changes in enzyme secondary structure. Analysis of cyanogen bromide fragments of 14C-carboxamidomethylated enzyme showed that all radioactivity was associated with cysteinyl residues in a single cyanogen bromide fragment. Three of these cysteinyl residues are clustered in a 38-residue region, which probably plays a role in maintaining the conformation of the kinase sugar phosphate-binding site.     

Water relations of the parasite: host relationship between the mistletoe Amyema linophyllum (Fenzl) Tieghem and Casuarina obesa Miq

Summary Seasonal and diurnal gas exchange and water relations of Amyema linophyllum and its host Casuarina obesa were studied at Gingin Western Australia. As recorded elsewhere for other species of mistletoe, stomatal conductances and transpiration rates were consistently higher in parasite than host, but assimilation rates did not differ significantly between partners, and water use efficiency was accordingly substantially lower in the parasite. Parallel responses of the species to environmental conditions suggested closely coordinated stomatal behaviour. However, sunlit and artifically shaded clumps of Amyema maintained high leaf conductances even when foliage fell below turgor loss point, yet their tissue capacitance values indicated maintenance of greater tissue water reserves during stress than in the host. Pressure-volume relationships indicated that differences in tissue water relations were unlikely to contribute significantly to the observed gradient in leaf water potential between partners. An experiment measuring changes in water potential of freshly detached host: parasite systems cut with the host shoot end immersed in water indicated that the haustorial junction was the principal site of resistance to transpiration-driven water flow into the parasite. A parallel experiment on intact attached shoots with mistletoe clumps enclosed and darkened just before dawn, demonstrated that, once the host commenced rapid transpiration, the water potential gradient between partners became reversed.     

Inhibition of nodule functioning in cowpea by a xanthine oxidoreductase inhibitor, allopurinol

Allopurinol (1H-pyrazolo-[3,4-d]pyrimidine-4-ol), an inhibitor of xanthine oxidation in ureide-producing nodulated legumes, was taken up from the rooting medium, translocated in xylem, and transferred to nodules of both the ureide-forming cowpea (Vigna unguiculata L. Walp.) and the amide-forming white lupin (Lupinus albus L.). Cowpea suffered severe nitrogen deficiency, extreme chlorosis, and reduced growth, whereas lupin was unaffected by the inhibitor. Similar results were obtained with oxypurinol (1H-pyrazolo-[3,4-d]pyrimidine-4,6-diol). Xylem composition of symbiotic cowpea was markedly changed by allopurinol. Ureides fell to a very low level, but xanthine and, to a lesser extent, hypoxanthine increased markedly. Xylem glutamine was also reduced, but there was little change in other amino acids. Nitrogenase (EC 1.7.99.2) activity of intact nodulated plants or nodulated root segments of plants treated with allopurinol or oxypurinol for 24 hours or more was severely inhibited in cowpea but unaffected in lupin for periods of exposure up to 9 days. Nitrogenase activity of slices of nodules prepared from allopurinol-treated cowpea showed inhibition comparable to that of intact plants. Breis prepared from nodules of treated plants showed no reduction in nitrogenase, nor was there reduction in activity of breis following addition of allopurinol, xanthine, or a range of purine pathway intermediates. Increasing the O₂ concentration in assays above 20% (volume/volume) reversed inhibition of nitrogenase by allopurinol in intact nodulated roots. It was concluded for cowpea that allopurinol not only inhibited ureide synthesis but also caused inhibition of nitrogenase activity, thereby leading to progressive dysfunction and eventual senescence of nodules. The mechanistic relationships between inhibition of ureide biosynthesis, changes in gaseous diffusion resistance, and reduced nitrogenase activity remain obscure.     

The utilization of nitrogen from insect capture by different growth forms of Drosera from Southwest Australia

Summary Plants of Drosera species, neighbouring noncarnivorous plants, and arthropods on or near each Drosera sp. were collected at 11 contrasting habitat locations in SW Australia. At three of the sites clones of the rare glandless mutant form of D. erythrorhiza were collected alongside fully glandular counterparts. The ¹⁵N value (¹⁵N/¹⁴N natural isotope composition) of insect-free leaf and stem fractions was measured, and the data then used to estimate proportional dependence on insect N (%N_dI) for the respective species and growth forms of Drosera. The data indicated lower %N_dI values for rosette than for self-supporting erect or for climbing vine species. The latter two groups showed an average %N_dI value close to 50%. The %N_dI increased with length and biomass of climbing but not erect forms of Drosera. ¹⁵N values of stems were positively correlated with corresponding values for leaves of Drosera. Leaf material was on average significantly more ¹⁵N enriched than stems, possibly due to delayed transport of recent insect-derived N, or to discrimination against ¹⁵N in transfer from leaf to the rest of the plant. The comparison of ¹⁵N values of insects and arthropod prey, glandless and glandular plants of D. erythrorhiza indicated %N_dI values of 14.3, 12.2 and 32.2 at the respective sites, while matching comparisons based on ¹⁵N of insect, reference plants and glandular plants proved less definitive, with only one site recording a positive %N_dI (value of 10.4%) despite evidence at all sites of feeding on insects by the glandular plants. The use of the ¹⁵N technique for studying nutrition of carnivorous species and the ecological significance of insect feeding of different growth forms of Drosera growing in a large range of habitats is discussed.     

A model for the interaction of muscle cross-bridges with ligands which compete with ATP

A model is presented to describe the inhibition of muscle fiber contraction by ligands that compete with MgATP. Two ligands, adenosine 5' (beta, gamma-imido) triphosphate (AMPPNP) and pyrophosphate (PPi), decrease the force developed in isometric contractions and act as weak competitive inhibitors of the maximum velocity of contraction (Pate & Cooke, 1985). These observations provide information on the energetics of actomyosin ligand states at the end of the power-stroke where MgATP dissociates the myosin cross-bridge from actin, and they are analysed in terms of a seven state model of cross-bridge kinetics. The model can reconcile the observations that these ligands bind tightly to fibers, Kd = 10(-4) M, while they are only weak inhibitors of fiber velocity, Ki = 2 X 10(-3) M. It provides a reasonable fit to the data and leads to several conclusions concerning the properties of the cross-bridge states. The states with bound ligand are shifted axially so that they occur earlier in the power-stroke than the nucleotide-free rigor state. This shift also explains the axial lengthening seen upon addition of ligands to rigor fibers. We can conclude that these ligands cause small perturbations in the cross-bridge configuration rather than large shifts. A second conclusion is that cross-bridges do not detach from actin during their power-strokes. Instead they traverse the entire length of the power stroke and are detached only at the end, leading to the suggestion that the cycling of bridges in isometric fibers is due to fluctuations in the relative positions of thick and thin filaments. With some further assumptions, the model also explains many of the rate constants and equilibrium constants of the actin-myosin-ligand interaction that have been measured in solution.     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N₂ fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

The inhibition of muscle contraction by adenosine 5' (beta, gamma-imido) triphosphate and by pyrophosphate.

We have studied the inhibition of the contraction of glycerinated rabbit psoas muscle caused by ligands that bind to the ATPase site of myosin. Two ligands, adenosine 5' (beta, gamma-imido) triphosphate (AMPPNP) and pyrophosphate (PPi), decreased the force and stiffness developed in isometric contractions and the velocity of shortening of isotonic contractions. The force exerted by isometric fibers was measured as a function of MgATP in the presence and absence of a constant concentration of the ligands. As the MgATP concentration decreased, the inhibition of tension caused by the ligand increased, reaching approximately 50% at 25 microM MgATP and either 2 mM MgPPi or 2 mM MgAMPPNP. The maximum velocity of shortening was also measured as a function of MgATP concentration in the presence of 1 and 2 mM MgPPi and 2.5 and 5 mM MgAMPPNP. Both ligands acted as pure competitive inhibitors with Ki = 3.0 mM for PPi and 5.1 mM for MgAMPPNP. These data show that both ligands are weak inhibitors of the contraction of fibers. The results provided information on the energetics of actin-myosin-ligand states that occur in the portion of the cross-bridge cycle where MgATP binds to myosin. A simple analysis of the inhibition of velocity suggests that MgAMPPNP binds to the actomyosin complex at this step of the cycle with an effective affinity constant of approximately 2 X 10(2) M-1.     

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.     

Significance of hydrogen evolution in the carbon and nitrogen economy of nodulated cowpea

The carbon and nitrogen economies of a single cultivar of cowpea (Vigna unguiculata (L.) Walp.cv Caloona) nodulated with either a high H₂-evolving strain (176A27) or a low H₂-evolving strain (CB756) of Rhizobium were compared. The two symbioses did not differ in total dry matter production, seed yield, nitrogen fixed, the spectrum of nitrogenous solutes produced by nodules for export, or the partitioning of net photosynthate within the plant throughout the growth cycle. Detailed examination of the carbon and nitrogen economy of the nodules, however, showed a significant difference between the symbioses. Nodules formed with CB756 lost less CO₂ in respiration compared to the higher H₂-evolving symbioses and this could have been largely responsible for a 36% better economy of carbon use in CB756 nodules during the period of maximum H₂ evolution (48-76 days) and over the whole growth period (20-90 days), a 16% economy. In terms of overall net photosynthate generated by the plant, these economies were equivalent to 5% and 2% of the carbon utilized in the two periods, respectively. From the differences in H₂ evolution and CO₂ production by nodules of the two symbioses, the cost of H₂ evolution was found to be 3.83±0.6 millimoles CO₂/millimoles H₂ for plants grown in sand culture and 1.69 ± 0.48 millimoles CO₂/millimoles H₂ for those in water culture. In both symbioses, the ratio of H₂ evolution to N₂ fixed varied markedly during ontogeny, indicating a significant variation in the relative efficiency and thus metabolic cost of N₂ fixation at different stages during development.