Cyril A. Appleby

Hemoglobin in a Nonleguminous Plant, Parasponia: Possible Genetic Origin and Function in Nitrogen Fixation

A dimeric hemoglobin was purified from nitrogen-fixing root nodules formed by association of Rhizobium with a nonleguminous plant, Parasponia. The oxygen dissociation rate constant is probably sufficiently high to allow Parasponia hemoglobin to function in a fashion similar to that of leghemoglobin, by oxygen buffering and transport during symbiotic nitrogen fixation. The identification of hemoglobin in a nonlegume raises important questions about the evolution of plant hemoglobin genes.

The purification, characterization and ligand-binding kinetics of hemoglobins from root nodules of the non-leguminous Casuarina glauca — Frankia symbiosis

The presence of a membrane-bound hemoglobin in aqueous extracts of nitrogen-fixing Casuarina root nodules (Davenport, H.E. (1960) Nature 186, 653–654) has been confirmed. By strictly anaerobic grinding and extraction under carbon monoxide, with inclusion of soluble polyvinylpyrrolidone and zwitterionic detergent in extraction buffer, soluble carboxyhemoglobin was obtained. This was purified by anaerobic ‘adsorption’ chromatography on Sephacryl S-200 (Pharmacia) followed by aerobic molecular exclusion chromatography on Sephadex G-75 (Pharmacia) to yield very stable oxyhemoglobin. By preparative-scale isoelectric focusing Casuarina oxyhemoglobin is separable into three major components comprising approx. 20% of applied protein, and very many minor components. Monomeric Casuarina hemoglobin is similar to other plant hemoglobins in respect of molecular weight (≈ 17 500), optical spectra, extremely rapid kinetics of binding to oxygen and carbon monoxide and high oxygen affinity (P50 ≈ 0.074 torr). Hence, it is possible that this protein functions in the Casuarina symbiosis as does leghemoglobin in leguminous nitrogen-fixing symbioses. Western blot analysis showed close immunological relationships between the non-leguminous Casuarina and Parasponia hemoglobins and a weaker relationship between these two proteins and soybean leghemoglobin. It is proposed that these hemoglobins from widely separated plant orders have a common evolutionary origin.

Studies of the physiological role of leghaemoglobin in soybean root nodules

Abstract Evolution of H2 by nitrogenase in intact soybean nodules was consistently inhibited by CO when the nodules were equilibrated with argon-CO mixtures for 1 h prior to adding O2 to initiate the reaction. Evolution of H2 by nitrogenase in bacteroid suspensions prepared from nodules, was not inhibited by CO. Dense, slowly shaken suspensions of bacteroids, with 12% O2 in the gas phase maintained slow rates of H2 evolution and acetylene reduction for up to 12 h. Addition of leghaemoglobin to these assays greatly enhanced the nitrogenase-mediated reactions. CO prevented stimulation by leghaemoglobin of H2 evolution by bacteroids. Stimulation of acetylene reduction by bacteroid suspensions was dependent upon leghaemoglobin concentration up to about 1 mM. Increased shaking rates gave greater rates of acetylene reduction and O2 uptake. Stimulation of nitrogenase activity in bacteroid suspensions by leghaemoglobin was much greater than stimulation of O2 uptake. Increasing acetylene reduction in response to increasing agitation was accompanied by increasing oxygenation of the leghaemoglobin. The significance of these results in relation to the physiological role of leghaemoglobin in symbiotic N2 fixation by legume root nodules is discussed.

Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants.

Hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa have been isolated [Landsmann et al. (1986). Nature 324, 166-168; Bogusz et al. (1988). Nature 331, 178-180]. The promoters of these genes have been linked to a beta-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus. Both promoters directed root-specific expression in transgenic tobacco. When transgenic Lotus plants were nodulated by Rhizobium loti, both promoter constructs showed a high level of nodule-specific expression confined to the central bacteroid-containing portion of the nodule corresponding to the expression seen for the endogenous Lotus leghemoglobin gene. The T. tomentosa promoter was also expressed at a low level in the vascular tissue of the Lotus roots. The hemoglobin promoters from both nonlegumes, including the non-nodulating species, must contain conserved cis-acting DNA signals that are responsible for nodule-specific expression in legumes. We have identified sequence motifs postulated previously as the nodule-specific regulatory elements of the soybean leghemoglobin genes [Stougaard et al. (1987). EMBO J. 6, 3565-3569].

Separation and determination of the relative concentrations of the homogeneous components of soybean leghemoglobin by isoelectric focusing.

The multiple components of soybean ferric leghemoglobin are readily separated by analytical and preparative flat bed isoelectric focusing in both the presence and also the absence of the ligand nicotinate. In the presence of nicotinate the separation by isoelectric focusing is more rapid and results in sharper bands of the very stable ferric leghemoglobin nicotinate complexes. The separation is sensitive enough to permit analytical experiments on leghemoglobin from single nodules. Leghemoglobins a and c1 prepared by ion exchange chromatography are homogeneous by isoelectric focusing criteria. Leghemoglobin c2 prepared by ion exchange chromatography is an approximately 1:2 mixture of leghemoglobins c2 and c3. Leghemoglobin d consists of three components. The ratio of leghemoglobin a to leghemoglobin c3 content increases dramatically as very young nodules mature. The increase in relative leghemoglobin a content suggests that leghemoglobin a might be required for regulation of nodule O2 concentration only when the nodule structure is complex. The ratio of leghemoglobin c1 content to leghemoglobin c3 content increases somewhat during the early period of nodule development, while the ratio of leghemoglobin c2 content to leghemoglobin c3 content increases slowly throughout nodule development. Ratios of leghemoglobin b content to leghemoglobin a content and of total leghemoglobin d content to total leghemoglobin c content were almost independent of nodule age. Leghemoglobins a and b might be related biosynthetically, as might leghemoglobins c and d.

Siderophore-bound iron in the peribacteriod space of soybean root nodules

Water-soluble, non-leghemoglobin iron (125 µmol kg-1 wet weight nodule) is found in extracts of soybean root nodules. This iron is probably confined to the peribacteroid space of the symbiosome, where its estimated concentration is 0.5 – 2.5 mM. This iron is bound by siderophores (compounds binding ferric iron strongly) which are different for each of the three strains of Bradyrhizobium japonicum with which the plants were inoculated. One of these, that from nodules inoculated with strain CC 705, is tentatively identified as a member of the pseudobactin family of siderophores.Leghemoglobin is present in only very small amounts in the peribacteroid space of symbiosomes isolated from soybean root nodules, and may be absent from the peribacteroid space of the intact nodule.

Amino acid sequence of hemoglobin I from root nodules of the non-leguminous Casuarina glauca-Frankia symbiosis

The amino acid sequence of hemoglobin I from nitrogen‐fixing root nodules of the Casuarina glauca‐Frankia symbiosis has been determined. The protein is composed of 151 amino acids including a single cysteine, a residue not found in the leghemoglobins. The molecular mass, including heme, is calculated to be 17856 Da. C. glauca hemoglobin I shows extensive sequence homology (43–52%) with other plant hemoglobins and this provides further evidence that hemoglobins from distant plant genera and animal hemoglobins share a common evolutionary origin.

Sesbania rostrata root and stem nodule leghemoglobins: purification, and relationships among the seven major components

By anion-exchange chromatography, the nitrogen fixing photosynthetic stem nodules and nonphotosynthetic root nodules of Sesbania rostrata are shown to contain the same seven major components of leghemoglobin (Lb), numbered LbI-LbVII in order of elution, although in different proportions. No novel component was found in photosynthetic nodules. All components of Sesbania Lb are monomeric, with molecular weights varying between 15,000 and 17,000, and at least six of them are separate gene products. It is suspected that variable conjugation with nonprotein moieties might be partially responsible for the molecular weight differences and anomalous behavior observed between isoelectric focusing and anion-exchange chromatography.

The amino acid sequence of hemoglobin I from Parasponia andersonii, a nonleguminous plant

The complete amino acid sequence of the hemoglobin I from nitrogen‐fixing root nodules of the nonleguminous plant, Parasponia andersonii, has been determined. This dimeric protein consists of two identical polypeptide chains of 155 amino acids and shows extensive sequence homology with other hemoglobins. Homology between the hemoglobin I of P. andersonii and the leghemoglobins of lupin and soybean nodules is 41 and 39%, respectively. The predicted secondary structure of P. andersonii hemoglobin I has a high content of α‐helix; except for the E‐helix, similar helices were predicted as those in the leghemoglobins. The close homology of the sequences provides evidence that this nonleguminous hemoglobin shares the same genetic origin as the legume and animal hemoglobins.

LEGHEMOGLOBIN: THE ROLE OF HEMOGLOBIN IN THE NITROGEN‐FIXING LEGUME ROOT NODULE*

A world increasingly short of protein may expend its limited supply of chemical or electrical energy t o fix atmospheric nitrogen into ammonia, or may rely in greater part on plants to harvest solar energy, some part of which is directed t o the support of bacterial nitrogen fixation. The rapid development of the nitrogen-fixing soybean as a major crop suggests that reliance on plants may already have become the more economical way t o regenerate the supply of fixed nitrogen. A hemeprotein, leghemoglobin, is an indispensable component of the system by which leguminous plants support the activity of nitrogen-fixing bacteria. We here inquire into the molecular mechanism by which leghemoglobin augments the oxygen consumption and nitrogen-fixing activity of bacteroids. That mechanism is leghemoglobin-facilitated oxygen diffusion brought about by translational diffusion of the oxygenated protein. The flux of oxygen is enhanced by facilitated diffusion. In addition, we now discover, facilitated diffusion makes oxygen more available to the terminal oxidases of subcellular organelles. We are concerned with the physicochemical definition of what we mean by “available.” Nitrogen-fixing nodules are formed on the roots of legumes in response t o invasion by bacteria of the genus Rhizobium. Rhizobia, modified for symbiotic life, are called bacteroids. The enzyme complex, nitrogenase, which is responsible for nitrogen fixation, is located wholly within the bacteroids. Nitrogenase does not utilize oxygen (in fact it is inhibited by even traces of oxygen) but depends for its activity of a supply of ATP formed (presumably) by bacteroidal oxidative phosphorylation. The bacteroids, which occur within the nodule cell, are in some ways analogous to muscle mitochondria. They occupy about the same fraction, approximately one-third,1,20f the cell volume and are responsible for the largest part of the oxygen consumption. Bacteroidal oxygen demand is vigorous; the oxygen consumption of the intact nodule is about one-tenth as great as that of the most active mammalian muscles. In fact, both