Nickel: A micronutrient element for hydrogen-dependent growth of \u3ci\u3eRhizobium japonicum\u3c/i\u3e and for expression of urease activity in soybean leaves

Soybean plants and Rhizobium japonicum 122 DES, a hydrogen uptake-positive strain, were cultured in media purified to remove Ni. Supplemental Ni had no significant effect on the dry matter or total N content of plants. However, the addition of Ni to both nitrate-grown and symbiotically grown plants resulted in a 7- to 10-fold increase in urease activity (urea amidohydrolase, EC 3.5.1.5) in leaves and significantly increased the hydrogenase activity (EC 1.18.3.1) in isolated nodule bacteroids. When cultured under chemolithotrophic conditions, free-living R. japonicum required Ni for growth and for the expression of hydrogenase activity. Hydrogenase activity was minimal or not detectable in cells incubated either without Ni or with Ni and chloramphenicol. Ni is required for derepression of hydrogenase activity and apparently protein synthesis is necessary for the participation of Ni in hydrogenase expression. The addition of Cr, V, Sn, and Pb in place of Ni failed to stimulate the activity of hydrogenase in R. japonicum and urease in soybean leaves. The evidence indicates that Ni is an important micronutrient element in the biology of the soybean plant and R. japonicum

Cloning and expression analysis of hemoglobin genes from maize (\u3ci\u3eZea mays\u3c/i\u3e ssp. \u3ci\u3emays\u3c/i\u3e) and teosinte (\u3ci\u3eZea mays\u3c/i\u3e ssp. \u3ci\u3eparviglumis\u3c/i\u3e)

With the exception of barley and rice, little is known about the existence of hemoglobins (Hbs) in cereals. This work reports the cloning and analysis of hb genes from maize (Zea mays ssp. mays) and teosinte (Zea mays ssp. parviglumis). Coding sequences of maize and teosinte hb genes (hbm and hbt, respectively) are highly similar to each other and are interrupted by three introns located at identical positions as other plant hb genes. Sequences of predicted Hbm and Hbt proteins are identical. The hydropathic profile of Hbm and Hbt is highly similar to that of rice Hb1, suggesting that Hbm, Hbt and Hb1 have the same tertiary structure and biochemical properties. Expression analysis showed that low levels of Hb transcripts, but considerable levels of Hb proteins exist in maize embryonic organs. No Hb transcripts and proteins were detected in teosinte embryonic organs. Low levels of Hb proteins, but no Hb transcripts, were detected in maize and teosinte vegetative organs. These observations suggest that the regulation of hb genes is different in maize and teosinte embryonic organs, and that the expression of hb genes is down- or up-regulated in maize and teosinte, respectively, from germination to vegetative growing

Cloning and expression analysis of hemoglobin genes from maize (\u3ci\u3eZea mays\u3c/i\u3e ssp. \u3ci\u3emays\u3c/i\u3e) and teosinte (\u3ci\u3eZea mays\u3c/i\u3e ssp. \u3ci\u3eparviglumis\u3c/i\u3e)

With the exception of barley and rice, little is known about the existence of hemoglobins (Hbs) in cereals. This work reports the cloning and analysis of hb genes from maize (Zea mays ssp. mays) and teosinte (Zea mays ssp. parviglumis). Coding sequences of maize and teosinte hb genes (hbm and hbt, respectively) are highly similar to each other and are interrupted by three introns located at identical positions as other plant hb genes. Sequences of predicted Hbm and Hbt proteins are identical. The hydropathic profile of Hbm and Hbt is highly similar to that of rice Hb1, suggesting that Hbm, Hbt and Hb1 have the same tertiary structure and biochemical properties. Expression analysis showed that low levels of Hb transcripts, but considerable levels of Hb proteins exist in maize embryonic organs. No Hb transcripts and proteins were detected in teosinte embryonic organs. Low levels of Hb proteins, but no Hb transcripts, were detected in maize and teosinte vegetative organs. These observations suggest that the regulation of hb genes is different in maize and teosinte embryonic organs, and that the expression of hb genes is down- or up-regulated in maize and teosinte, respectively, from germination to vegetative growing

Crystal Structure of a Nonsymbiotic Plant Hemoglobin

Background: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O2 conditions. Results: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 Å resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b5. The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. Conclusions: The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O2 reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O2 affinity is ultra-high (KD ≈ 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified

Enzymatic and nonenzymatic mechanisms for ferric leghemoglobin reduction in legume root nodules

Evidence is presented for the operation in nodules of at least four systems for restoring functional ferrous leghemoglobin (Lb2+) from its inactive, ferric form. (i) Reduction of ferric leghemoglobin (Lb3+) by a reductase. The enzyme is a flavoprotein of 100 kDa with two equally sized subunits and exhibits a Km of 9 µM for soybean Lb3+ component a and a K. of 51 µM for NADH. NADPH is only 30% (initial velocities) as effective as NADH. Lb3+ reductase converts 215 nmol of Lb3+to Lb2+ •CO (or Lb2+ •O2) per mg of protein per min and does not require an exogenous electron carrier. The enzyme shows similar affinity for soybean, bean, and cowpea Lb3+, but different Vmax values. The reductase is inactive when Lb3+ is bound to nicotinate or N02 ˉ. (ii) Direct reduction of Lb3+ by NAD(P)H, ascorbate, and cysteine. Reduction by NAD(P)H is greatly stimulated by trace amounts of metals such as Mn2+. (iii) Reduction of Lb3+ by the flow of electrons from NAD(P)H to free flavins to Lb3+. The reaction does not occur via 02ˉ or H202, and thus NAD(P)H-reduced flavins can directly reduce Lb3+. The efficiency of the reaction follows the order riboflavin \u3e FMN \u3e FAD. (iv) Reduction of Lb3+ by an unknown compound, B, of nodules. B has a molecular mass \u3c 1 kDa and is heat-stable. The reaction mediated by B differs from those mediated by flavins and metals in several ways, requires NAD(P)H, and generates 02ˉ