Molecular cloning and characterization of a moss (Ceratodon purpureus) nonsymbiotic hemoglobin provides insight into the early evolution of plant nonsymbiotic hemoglobins

Nonsymbiotic hemoglobins (nsHbs) are widespread in plants including bryophytes. Bryophytes (such as mosses) are among the oldest land plants, thus an analysis of a bryophyte nsHb is of interest from an evolutionary perspective. However, very little is known about bryophyte nsHbs. Here, we report the cloning and characterization of an nshb gene (cerhb) from the moss Ceratodon purpureus. Sequence analysis showed that cerhb is interrupted by 3 introns in identical position as all known plant nshb genes, which suggests that the ancestral nshb gene was interrupted by 3 introns. Expression analysis showed that cerhb expresses in protonemas and gametophytes growing in normal conditions and that it overexpresses in protonemas subjected to osmotic (sucrose), heat-shock, cold-, and nitrate-stress conditions. Also, modeling of the Ceratodon nsHb (CerHb) tertiary structure suggests that CerHb is hexacoordinate and that it binds O(2) with high affinity. Comparative analysis of the predicted CerHb with native rice Hb1 and soybean leghemoglobin a structures revealed that the major evolutionary changes that probably occurred during the evolution of plant Hbs were 1) a hexacoordinate to pentacoordinate transition at the heme prosthetic group, 2) a length decrease at the CD-loop and N- and C-termini regions, and 3) the compaction of the protein into a globular structure.     

Prediction of folding pathway and kinetics among plant hemoglobins using an average distance map method

Computational methods, such as the ADM (average distance map) method, have been developed to predict folding of homologous proteins. In this work we used the ADM method to predict the folding pathway and kinetics among selected plant nonsymbiotic (nsHb), symbiotic (Lb), and truncated (tHb) hemoglobins (Hbs). Results predicted that (1) folding of plant Hbs occurs throughout the formation of compact folding modules mostly formed by helices A, B, and C, and E, F, G, and H (folding modules A/C and E/H, respectively), and (2) primitive (moss) nsHbs fold in the C-->N direction, evolved (monocot and dicot) nsHbs fold either in the C-->N or N-->C direction, and Lbs and plant tHbs fold in the C-->N direction. We also predicted relative folding rates of plant Hbs from qualitative analyses of the stability of subdomains and classified plant Hbs into fast and moderate folding. ADM analysis of nsHbs predicted that prehelix A plays a role during folding of the N-terminal domain of Ceratodon nsHb, and that CD-loop plays a role in folding of primitive (Physcomitrella and Ceratodon) but not evolved nsHbs. Modeling of the rice Hb1 A/C and E/H modules showed that module E/H overlaps to the Mycobacterium tuberculosis HbO two-on-two folding. This observation suggests that module E/H is an ancient tertiary structure in plant Hbs.     

A model for ligand binding to hexacoordinate hemoglobins

Hexacoordinate hemoglobins are heme proteins capable of reversible intramolecular coordination of the ligand binding site by an amino acid side chain from within the heme pocket. Examples of these proteins are found in many living organisms ranging from prokaryotes to humans. The nonsymbiotic hemoglobins (nsHbs) are a class of hexacoordinate heme proteins present in all plants. The nsHb from rice (rHb1) has been used as a model system to develop methods for determining rate constants characterizing binding and dissociation of the His residue responsible for hexacoordination. Measurement of these reactions exploits laser flash photolysis to initiate the reaction from the unligated, pentacoordinate form of the heme protein. A model for ligand binding is presented that incorporates the reaction following rapid mixing with the reaction starting from the pentacoordinate hemoglobin (Hb). This model is based on results indicating that ligand binding to hexacoordinate Hbs is not a simple combination of competing first order (hexacoordination) and second order (exogenous ligand binding) reactions. Ligand binding following rapid mixing is a multiphasic reaction displaying time courses ranging from milliseconds to minutes. The new model incorporates a "closed", slow reacting form of the protein that is not at rapid equilibrium with the reactive conformation. It is also demonstrated that formation of the closed protein species is not dependent on hexacoordination.     

A JAZ Protein in Astragalus sinicus Interacts with a Leghemoglobin through the TIFY Domain and Is Involved in Nodule Development and Nitrogen Fixation

Leghemoglobins (Lbs) play an important role in legumes-rhizobia symbiosis. Lbs bind O₂ and protect nitrogenase activity from damage by O₂ in nodules, therefore, they are regarded as a marker of active nitrogen fixation in nodules. Additionally, Lbs are involved in the nitric oxide (NO) signaling pathway, acting as a NO scavenger during nodule development and nitrogen fixation. However, regulators responsible for Lb expression and modulation of Lb activity have not been characterized. In our previous work, a Jasmonate-Zim-domain (JAZ) protein interacting with a Lb (AsB2510) in Astragalus sinicus was identified and designated AsJAZ1. In this study, the interaction between AsJAZ1 and AsB2510 was verified using a yeast two-hybrid system and in vitro Glutathione S-transferase (GST) pull-down assays, resulting in identification of the interaction domain as a TIFY (previously known as zinc-finger protein expressed in inflorescence meristem, ZIM) domain. TIFY domain is named after the most conserved amino acids within the domain. Bimolecular fluorescence complementation (BiFC) was used to confirm the interaction between AsJAZ1 and AsB2510 in tobacco cells, demonstrating that AsJAZ1-AsB2510 interaction was localized to the cell membrane and cytoplasm. Furthermore, the expression patterns and the symbiotic phenotypes of AsJAZ1 were investigated. Knockdown of AsJAZ1 expression via RNA interference led to decreased number of nodules, abnormal development of bacteroids, accumulation of poly-x-hydroxybutyrate (PHB) and loss of nitrogenase activity. Taken together, our results suggest that AsJAZ1 interacts with AsB2510 and participates in nodule development and nitrogen fixation. Our results provide novel insights into the functions of Lbs or JAZ proteins during legume-rhizobia symbiosis.     

Nitric oxide detoxification in the rhizobia-legume symbiosis

NO (nitric oxide) is a signal molecule involved in diverse physiological processes in cells which can become very toxic under certain conditions determined by its rate of production and diffusion. Several studies have clearly shown the production of NO in early stages of rhizobia-legume symbiosis and in mature nodules. In functioning nodules, it has been demonstrated that NO, which has been reported as a potent inhibitor of nitrogenase activity, can bind Lb (leghaemoglobin) to form LbNOs (nitrosyl-leghaemoglobin complexes). These observations have led to the question of how nodules overcome the toxicity of NO. On the bacterial side, one candidate for NO detoxification in nodules is the respiratory Nor (NO reductase) that catalyses the reduction of NO to nitrous oxide. In addition, rhizobial fHbs (flavohaemoglobins) and single-domain Hbs which dioxygenate NO to form nitrate are candidates to detoxify NO under free-living and symbiotic conditions. On the plant side, sHbs (symbiotic Hbs) (Lb) and nsHbs (non-symbiotic Hbs) have been proposed to play important roles as modulators of NO levels in the rhizobia-legume symbiosis. In the present review, current knowledge of NO detoxification by legume-associated endosymbiotic bacteria is summarized.     

Quaternary structure effects on the hexacoordination equilibrium in rice hemoglobin rHb1: Insights from molecular dynamics simulations

Nonsymbiotic hemoglobins (nsHbs) form a widely distributed class of plant proteins, which function remains unknown. Despite the fact that class 1 plant nonsymbiotic hemoglobins are hexacoordinate (6c) heme proteins (hxHbs), their hexacoordination equilibrium constants are much lower than in hxHbs from animals or bacteria. In addition, they are characterized by having very high oxygen affinities and low oxygen dissociation rate constants. Rice hemoglobin 1 (rHb1) is a class 1 nonsymbiotic hemoglobin. It crystallizes as a fully associated homodimer with both subunits in 6c state, but showing slightly different conformations, thus leading to an asymmetric crystallographic homodimer. The residues that constitute the dimeric interface are conserved among all nsHbs, suggesting that the quaternary structure could be relevant to explain the chemical behavior and biological function of this family of proteins. In this work, we analyze the molecular basis that determine the hexacoordination equilibrium in rHb1. Our results indicate that dynamical features of the quaternary structure significantly affect the hexacoordination process. Specifically, we observe that the pentacoordinate state is stabilized in the dimer with respect to the isolated monomers. Moreover, the dimer behaves asymmetrically, in a negative cooperative scheme. The results presented in this work are fully consistent with our previous hypothesis about the key role played by the nature of the CD region in determining the coordination state of globins. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

An assessment of the biotechnological use of hemoglobin modulation in cereals

Non‐symbiotic hemoglobin (nsHb) genes are ubiquitous in plants, but their biological functions have mostly been studied in model plant species rather than in crops. nsHb influences cell signaling and metabolism by modulating the levels of nitric oxide (NO). Class 1 nsHb is upregulated under hypoxia and is involved in various biotic and abiotic stress responses. Ectopic overexpression of nsHb in Arabidopsis thaliana accelerates development, whilst targeted overexpression in seeds can increase seed yield. Such observations suggest that manipulating nsHb could be a valid biotechnological target. We studied the effects of overexpression of class 1 nsHb in the monocotyledonous crop plant barley (Hordeum vulgare cv. Golden Promise). nsHb was shown to be involved in NO metabolism in barley, as ectopic overexpression reduced the amount of NO released during hypoxia. Further, as in Arabidopsis, nsHb overexpression compromised basal resistance toward pathogens in barley. However, unlike Arabidopsis, nsHb ectopic overexpression delayed growth and development in barley, and seed specific overexpression reduced seed yield. Thus, nsHb overexpression in barley does not seem to be an efficient strategy for increasing yield in cereal crops. These findings highlight the necessity for using actual crop plants rather than laboratory model plants when assessing the effects of biotechnological approaches to crop improvement.     

Catalase Activity of Nodules in the Ureide- and Amide-transporting Legume Plants

The relations of catalase activity to the efficiency of symbiotic dinitrogen fixation and leghemoglobin (Lb) content were investigated in roots and nodules of several legume plant species together with the catalase distribution between the inner bacteroidal and the outer cortical nodule tissues. The catalase activity in the nodules exceeded that of the roots of the amide- and ureide-synthesizing plant species by one and two orders of magnitude. During the growth period, catalase activity and Lb content changed in parallel and reached their highest levels early in the stage of flowering or fruit formation, depending on plant species. In the case of effective symbiosis, catalase activity in the nodules was 2.5–5 times higher than in the case of ineffective symbiosis. Catalase activity in the bacteroidal zone of the nodules was several times higher than that of the cortical tissue, and two nodule tissues differed in catalase activity more notably in the plant species exporting ureides. The authors suggest that high catalase activity in the nodules, especially in their bacteroidal zone, is essential for the efficient functioning of the symbiotic system of dinitrogen fixation in both ureide- and amide-transporting plants.     

Symbiotic and nonsymbiotic hemoglobin genes of Casuarina glauca.

Casuarina glauca has a gene encoding hemoglobin (cashb-nonsym). This gene is expressed in a number of plant tissues. Casuarina also has a second family of hemoglobin genes (cashb-sym) expressed at a high level in the nodules that Casuarina forms in a nitrogen-fixing symbiosis with the actinomycete Frankia. Both the nonsymbiotic and symbiotic genes retained their specific patterns of expression when introduced into the legume Lotus corniculatus. We interpret this finding to mean that the controls of expression of the symbiotic gene in Casuarina must be similar to the controls of expression of the leghemoglobin genes that operate in nodules formed during the interaction between rhizobia and legumes. Deletion analyses of the promoters of the Casuarina symbiotic genes delineated a region that contains nodulin motifs identified in legumes; this region is critical for the controlled expression of the Casuarina gene. The finding that the nonsymbiotic Casuarina gene is also correctly expressed in L. corniculatus suggests to us that a comparable non-symbiotic hemoglobin gene will be found in legume species.     

Molecular cloning and characterization of a nonsymbiotic hemoglobin gene (GLB1) from Malus hupehensis Rehd. with heterologous expression in tomato

Nonsymbiotic hemoglobins (nsHbs) are involved in a variety of cellular processes in plants. Previous studies indicate that nsHb expression improves plant tolerance during waterlogging and hypoxia. In the present work, the nsHb class-1 coding sequence was cloned from Malus hupehensis Rehd. var. pinyiensis Jiang and subsequently named MhGLB1. The results elucidated the expressed characteristics and physiological effects of MhGLB1. The full-length cDNA contained a 477 bp open reading frame encoding a protein with a molecular mass of 17.8 KDa with 158 amino acids. Quantitative real-time PCR analysis showed that MhGLB1 expresses in roots, stems and leaves growing under normal and nitrate-induced conditions. Hypoxic stress induced accumulation of MhGLB1 within 12 h, and abscisic acid significantly induced expression of MhGLB1 in roots. The photosynthetic, transpiration and stomatal conductance rates of transgenic MhGLB1 tomato plants decreased more slowly than that of wild-type plants under waterlogging treatment. These results indicated that the MhGLB1 gene has an important role in hypoxia.     

Plant hemoglobins: important players at the crossroads between oxygen and nitric oxide

Plant hemoglobins constitute a diverse group of hemeproteins and evolutionarily belong to three different classes. Class 1 hemoglobins possess an extremely high affinity to oxygen and their main function consists in scavenging of nitric oxide (NO) at very low oxygen levels. Class 2 hemoglobins have a lower oxygen affinity and they facilitate oxygen supply to developing tissues. Symbiotic hemoglobins in nodules have mostly evolved from class 2 hemoglobins. Class 3 hemoglobins are truncated and represent a clade with a very low similarity to class 1 and 2 hemoglobins. They may regulate oxygen delivery at high O₂ concentrations. Depending on their physical properties, hemoglobins belong either to hexacoordinate non-symbiotic or pentacoordinate symbiotic groups. Plant hemoglobins are plausible targets for improving resistance to multiple stresses.     

Changes in four leghemoglobin components in nodules of hypernodulating soybean (Glycine max [L] Merr.) mutant and its parent in the early nodule developmental stage

Soybean nodules contain four major leghemoglobin (Lb) components, Lba, Lbc₁, Lbc₂ and Lbc₃. A sensitive and selective method for quantitative analysis of the four Lb components was devised with capillary isoelectric focusing (CIEF). The changes in the concentrations of four Lb components in nodules during the initial stages of development were compared between hypernodulating soybean mutant NOD1–3 and its parent cv. Williams. The hydroponically cultivated soybean plants were periodically sampled. All the visible nodules were collected from the roots, and then the four Lb components in the largest nodules were analyzed with the CIEF method. In NOD1–3 Lbs were initially detected at 19 days after sowing (DAS), a few days earlier than in Williams at 22 DAS. The Lbcs (Lbc₁, Lbc₂ and Lbc₃) were the main component at the earliest nodule growth stage, and the relative proportion of Lba increased with nodule growth in both NOD1–3 and Williams. This result is in agreement with previous observation, and the CIEF method is considered to be useful for Lb components analysis to define their function and gene expression.     

Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport

The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport "leghemoglobins" evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.     

A Re-Evaluation of the Role of the Infected Cell in the Control of O2 Diffusion in Legume Nodules

Two different simulation models were constructed to describe O2 diffusion into the bacteria-infected cells of legume nodules: one based on a central zone of uniform spherical cells and the other on a central zone of packed, uniform cubical cells with air spaces along the edges. The cubical model more closely approximated the geometry and gas diffusion characteristics of infected cells than did the spherical model. The models relied on set values for the innermost O2 concentration in the infected cell (1-20 nM) and predicted values for the free O2 and oxygenated leghemoglobin gradients toward the cell:space interface. The cubical model but not the spherical model predicted saturation of leghemoglobin (Lb) oxygenation at or within a few micrometers of the gas-filled intercellular space and predicted that the space concentration could be as high as 1.3% O2 when the fractional oxygenation of Lb and respiration rate within the infected cell were typical of that which has been measured in vivo. In the model, the higher the space O2 concentration, the greater the saturation of Lb by O2 and the greater the collapse of Lb-facilitated diffusion near the cell:space interface. This was predicted to result in a greater resistance to O2 diffusion from the space to the bacteroids, thereby providing an intrinsic, homeostatic mechanism for controlling the rate of O2 influx into infected cells. Changes in the physiological features of the simulated cubical infected cell, such as the proportion of the cell as cytosol, the surface area of the cell exposed to a space, the maximum rate of cellular respiration, or the concentration of Lb in the cytoplasm, significantly altered the extent to which the infected cell would be able to regulate its diffusive resistance. These results demonstrate the possibility of a Lb-based mechanism for controlling the O2 concentration within the infected cells. If such a mechanism exists in legume nodules, it would give the infected cell an ability to exercise fine control over its internal environment, a process that could complement a physical diffusion barrier that may exist in the inner cortex or elsewhere in the nodule and provide coarse control over O2 diffusion.     

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

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

Developmental regulation of nodule-specific genes in alfalfa root nodules

We have cloned alfalfa nodule-specific cDNAs that code for leghemoglobin (Lb), glutamine synthetase (GS), and three unidentified nodulins. Hybrid-select translation of nodule RNA followed by 2-D gel electrophoresis showed that the Lb-specific cDNA corresponded to at least four Lb species of 12 kDa. One of the unidentified cDNA clones (N-32/34) corresponded to at least five polypeptides of 32-34 kDa; a second unidentified cDNA clone (N-14) corresponded to an individual polypeptide of 14 kDa. The in vitro translation product(s) of the RNA hybrid selected by the third unidentified cDNA clone (N-22) formed a single band at 22 kDa on a one-dimensional gel. Northern and dot blot analyses of RNA isolated from wild-type nodules and from defective nodules elicited by a variety of Rhizobium meliloti mutants showed that 1) RNAs corresponding to the Lb, nodule-specific GS, and three unidentified nodulins were coordinately expressed during the course of nodule development, and 2) all five nodulins were expressed in Fix- nodules that contained infection threads and bacteroids but were not expressed in nodules that lacked infection threads and intracellular rhizobia.     

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 O(2) conditions. RESULTS: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 A 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 b(5). 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 O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 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.