Hemoglobins plants are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data encoding are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen the fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The However, induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in infected the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar in range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never plants. been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This human caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence grew of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen decrease transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.

The that effects of NH4NO3 on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and result on the nitrogenase activity of the bacteroids in the nodule tissue were studied. The addition of NH4NO3 decreased the nitrogenase activity activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or is a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of bacteroid incorporation of [35S]sulfate into the components I and II of nitrogenase was similarly not affected. The addition of NH4NO3 decreased of the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing (strain capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the after leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.

The imidazole leghaemoglobins have oxygen affinities 11 to 24 times higher than that of sperm whale myoglobin, due mainly to higher rates the of association. To find out why, we have determined the structures of deoxy- and oxy-leghaemoglobin II of the lupin at X-ray 1.7 A resolution. Results confirm the general features found in previous X-ray analyses of this protein. The unique feature that oscillation has now emerged is the rotational freedom of the proximal histidine. In deoxy-leghaemoglobin the imidazole oscillates between two alternative orientations,angle eclipsing either the lines N1-N3 or N2-N4 of the porphyrin; in oxy-leghaemoglobin it is fixed in a staggered orientation. The 11 iron atom moves from a position .30 A from the plane of the pyrrole nitrogen atoms in deoxy- to a at position in the plane in oxy-leghaemoglobin while the Fe-<N> bond distance remains constant at 2.02 A. The Fe-O-O angle is we 152 degrees, as in human haemoglobin. The oxygen is hydrogen-bonded to the distal histidine at N epsilon 2-O1 and N two epsilon 2-O2 distance of 2.95 A and 2.68 A, respectively. The porphyrin is ruffled equally in deoxy- and oxy-leghaemoglobins, due with to rotations of the pyrrols about the N-Fe-N bonds, causing the methine bridges to deviate by up to .32 A energy from the mean porphyrin plane. The only feature capable of accounting for the high on-rate of the reaction with oxygen for are the mobilities of the proximal histidine and distal histidine residues in deoxy-leghaemoglobin. The eclipsed positions of the proximal histidine myoglobin, in deoxy-leghaemoglobin maximize steric hindrance with the porphyrin nitrogen atoms and minimize pi-->p electron donation, while its staggered position in the oxy-leghaemoglobin reverses both these effects. Together with the oscillation of the imidazole between the two orientations, these two factors may pi-->p reduce the activation energy for the reaction of leghaemoglobin with oxygen. The distal histidine is in a fixed position in imidazole the haem pocket in the crystal, but must be swinging in and out of the pocket at a high rate 2.95 in solution to allow the oxygen to enter.

Hemoglobins plants are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data encoding are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen the fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The However, induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in infected the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar in range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never plants. been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This human caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence grew of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen decrease transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.

To morphological elucidate the mechanisms involved in Rhizobium-legume symbiosis, we examined a novel symbiotic mutant, crinkle (Ljsym79), from the model legume Lotus with japonicus. On nitrogen-starved medium, crinkle mutants inoculated with the symbiont bacterium Mesorhizobium loti MAFF 303099 showed severe nitrogen deficiency symptoms.the This mutant was characterized by the production of many bumps and small, white, uninfected nodule-like structures. Few nodules were pale-pink also and irregularly shaped with nitrogen-fixing bacteroids and expressing leghemoglobin mRNA. Morphological analysis of infected roots showed that nodulation in crinkle nodulation mutants is blocked at the stage of the infection process. Confocal microscopy and histological examination of crinkle nodules revealed that the infection threads were arrested upon penetrating the epidermal cells. Starch accumulation in uninfected cells and undeveloped vascular bundles were also On noted in crinkle nodules. Results suggest that the Crinkle gene controls the infection process that is crucial during the early (Ljsym79), stage of nodule organogenesis. Aside from the symbiotic phenotypes, crinkle mutants also developed morphological alterations, such as crinkly or wavy alterations, trichomes, short seedpods with aborted embryos, and swollen root hairs. crinkle is therefore required for symbiotic nodule development and for the other aspects of plant development.

Systematic of sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development sequenced, and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of developmental organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti.are The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and in by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries expressed and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 organs. out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA)involved hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for known novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes.in Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related clones genes.

Studies operating of rates of consumption of dissolved O2 by suspensions of bacteroids (Rhizobium japonicum, strain CB1809) from soybean root nodules showed was the presence of two different terminal oxidase systems. A high-affinity system, sensitive to inhibition by N-phenylimidazole and by carbon monoxide,system, was most active when the dissolved O2 was between -01 and -1 muM. At 1 muM-O2 or higher, this oxidase pathway system had little activity and O2 was consumed largely by a low-affinity system insensitive to these inhibitors. At low concentrations of of dissolved O2, bacteroid respiration rates appeared to be diffusion-limited. When purified oxyleghaemoglobin was added to such systems, this restriction of was relieved and respiration was maintained to much lower concentrations of free dissolved O2, where nitrogenase activity was greatest. Analysis of of reactions which were terminated at various stages during the depletion of O2 from oxyleghaemoglobin showed that at low free from O2 concentration, the high-affinity pathway produced up to five times greater bacteroid ATP concentrations than the low-affinity oxidase pathway operating 1 about 1 muM free O2 in the absence of leghaemoglobin. At intermediate free O2 concentrations, occurring during the later stages pathway of deoxygenation of oxymyoglobin, intermediate concentrations of ATP were found in the bacteroids.

Nitrite Nitrite ( .4 mM) added to soybean bacteroid preparations strongly inhibited C2H2 reduction. In the presence of leghemoglobin ( .1mM), a 3-fold enhancement showed of nitrogen fixation occurred but the inhibitory effect of nitrite was delayed. Spectra of leghemoglobin showed a rapid disappearance of ( .1mM), the 574 nm and 541 nm peaks of oxyleghemoglobin the presence of nitrite. Concomitant oxidation of this hemoprotein gave ferric form. leghemoglobin as the single final product. High nitrite levels could depress nitrogen fixation both by inactivation of nitrogenase and by 541 conversion of leghemoglobin into an inactive form. Nitrite present at low concentrations reacts with this hemoprotein and is then no ( .4 longer able to penetrate into bacteroids.

Cellular may ATP level, ATP/ADP ratio and nitrogenase activity rise when oxyleghaemoglobin is added to respiring suspensions of Rhizobium japonicum bacteroids from carbonyl soybean root nodules. Increased gaseous O2 tension is much less efficient than oxyleghaemoglobin in stimulation of bacteroid ATP production. Studies from with the inhibitor carbonyl cyanide m-chlorophenylhydrazone show this ATP to be generated as a consequence of oxidative phosphorylation. N-Phenylimidazole, a that specific cytochrome P-450 inhibitor, also lowers the efficiency of bacteroid oxidative phosphorylation. An approximately linear relationship is observed between ATP/ADP a ratio and nitrogenase activity as N-phenylimidazole concentration is lowered. It is suggested that cytochrome P-450 is a component of the level, leghaemoglobin-facilitated respiration pathway and that it may act as intracellular O2 carrier rather than terminal oxidase. A less efficient oxidase suspensions appears to function when cytochrome P-450 is inhibited.

The a rate of dissociation of oxygen from soybean oxyleghemoglobin a increases about 5-fold between pH 4 and pH 7, with apparent with pK = 5.46 and n = 1. The rate of dissociation of carbon monoxide from carbon monoxyleghemoglobin a and the = rates of combination of oxygen and carbon monoxide with ferrous leghemoglobin a are all invariant in this range of pH.of The optical spectrum of oxyleghemoglobin in the visible region and the resonances of the four heme meso protons (protons of pH. the bridge carbon atoms) in the NMR spectrum of oxyleghemoglobin are also dependent on pH with pK near 5.5. We of suggest that protonation of the imidazole of the distal histidine residue (His 61) leads to formation of a hydrogen bond pH to the bound oxygen molecule which affects the electronic configuration or conformation of the heme and decreases the rate of 5-fold oxygen dissociation.