Plant Haemoglobins,Nitric Oxide and Hypoxic Stress

It is now known that there are several classes of haemoglobins in plants. A specialized class of haemoglobins, symbiotic haemoglobins, were discovered 62 years ago and are found only in nodules of plants capable of symbiotic nitrogen fixation. Plant haemoglobins, with properties distinct from symbiotic haemoglobins were discovered 18 years ago and are now believed to exist throughout the plant kingdom. They are expressed in different organs and tissues of both dicot and monocot plants. They are induced by hypoxic stress and by oversupply of certain nutrients. Most recently, truncated haemoglobins have been shown to also exist in plants. While hypoxic stress‐induced haemoglobins are widespread in the plant kingdom, their function has not been elucidated. This review discusses the recent findings regarding the function of these haemoglobins in relation to adaptation to hypoxia in plants. We propose that nitric oxide is an important metabolite in hypoxic plant cells and that at least one of the functions of hypoxic stress‐induced haemoglobins is to modulate nitric oxide levels in the cell.     

Expression and evolution of functionally distinct haemoglobin genes in plants

Haemoglobin genes have been found in a number of plant species, but the number of genes known has been too small to allow effective evolutionary inferences. We present nine new non-symbiotic haemoglobin sequences from a range of plants, including class 1 haemoglobins from cotton, Citrus and tomato, class 2 haemoglobins from cotton, tomato, sugar beet and canola and two haemoglobins from the non-vascular plants, Marchantia polymorpha (a liverwort) and Physcomitrella patens (a moss). Our molecular phylogenetic analysis of all currently known non-symbiotic haemoglobin genes and a selection of symbiotic haemoglobins have confirmed the existence of two distinct classes of haemoglobin genes in the dicots. It is likely that all dicots have both class 1 and class 2 non-symbiotic haemoglobin genes whereas in monocots we have detected only class 1 genes. The symbiotic haemoglobins from legumes and Casuarina are related to the class 2 non-symbiotic haemoglobins, whilst the symbiotic haemoglobin from Parasponia groups with the class 1 non-symbiotic genes. Probably, there have been two independent recruitments of symbiotic haemoglobins. Although the functions of the two non-symbiotic haemoglobins remain unknown, their patterns of expression within plants suggest different functions. We examined the expression in transgenic plants of the two non-symbiotic haemoglobins from Arabidopsis using promoter fusions to a GUS reporter gene. The Arabidopsis GLB1 and GLB2 genes are likely to be functionally distinct. The class 2 haemoglobin gene (GLB2) is expressed in the roots, leaves and inflorescence and can be induced in young plants by cytokinin treatment in contrast to the class 1 gene (GLB1) which is active in germinating seedlings and can be induced by hypoxia and increased sucrose supply, but not by cytokinin treatment.     

Multiple globins and haemoglobins in the bass, Dicentrarchus labrax (L.) (Serranidae: Teleostei)

The haemoglobins and globins of bass, Dicentrarchus labrax (L.) have been studied by starch and polyacrylamide gel electrophoresis. Five haemoglobin components were found. These haemoglobins appear to result from the combination of four different globin monomers. The molecular weight of the pooled haemoglobin is about 54 400, confirming its tetrameric form. The evolutionary significance of multiple haemoglobins is discussed.     

Structural and functional analysis of the two haemoglobins of the antarctic seabird Catharacta maccormicki characterization of an additional phosphate binding site by molecular modelling.

The amino-acid sequence and the oxygen-binding properties of the two haemoglobins of the Antarctic seabird south polar skua have been investigated. The two haemoglobins showed peculiar functional features, which were probably acquired to meet special needs in relation to the extreme environmental conditions. Both haemoglobins showed a weak alkaline Bohr effect which, during prolonged flight, may protect against sudden and uncontrolled stripping of oxygen in response to acidosis. We suggest that a weak Bohr effect in birds may reflect adaptation to extreme life conditions. The values of heat of oxygenation suggest different functional roles of the two haemoglobins. The experimental evidence suggests that both haemoglobins may bind phosphate at two distinct binding sites. In fact, analysis of the molecular models revealed that an additional phosphate binding site, formed by residues NA1alpha, G6alpha and HC3alpha, is located between the two alpha chains. This additional site may act as an entry/leaving site, thus increasing the probability of capturing phosphate and transferring it to the main binding site located between the two beta chains by means of a site-site migratory mechanism, thereby favouring the release of oxygen. It is suggested that most haemoglobins possess an additional phosphate binding site, having such a role in oxygen transport.     

Evolutionary trace analysis of plant haemoglobins: implications for site-directed mutagenesis

Haemoglobins are found ubiquitously in eukaryotes and many bacteria. In plants, haemoglobins were first identified in species, which can fix nitrogen via symbiosis with bacteria. Recent findings suggest that another class of haemoglobins termed as nonsymbiotic haemoglobins are present through out the plant kingdom and are expressed differentially during plant development. Limited data available suggests that non-symbiotic haemoglobins are involved in hypoxic stress and oversupply of nutrients. Due to lack of information on structurally conserved, functionally important residues in non-symbiotic haemoglobins, further studies to elucidate the molecular mechanisms underlying the biological role are hampered. To determine functionally important residues in non-symbiotic haemoglobins, I have analyzed a number of sequences from plant haemoglobin family, in the context of the known crystal structures of plant by evolutionary trace method. Results indicate that the, evolutionary trace method like conventional phylogentic analysis, could resolve phylogentic relationships between plant haemoglobin family. Evolutionary trace analysis has identified candidate functional (trace) residues that uniquely characterize the heme-binding pocket, dimer interface and possible novel functional surfaces. Such residues from specific three-dimensional clusters might be of functional importance in nonsymbiotic haemoglobins. These data, together with our improved knowledge of possible functional residues, can be used in future structure-function analysis experiments.     

Primary structure of a constituent polypeptide chain (AIII) of the giant haemoglobin from the deep-sea tube worm Lamellibrachia. A possible H2S-binding site.

The deep-sea tube worm Lamellibrachia, belonging to the Phylum Vestimentifera, contains two giant extracellular haemoglobins, a 3000 kDa haemoglobin and a 440 kDa haemoglobin. The former consists of four haem-containing chains (AI-AIV) and two linker chains (AV and AVI) for the assembly of the haem-containing chains [Suzuki, Takagi & Ohta (1988) Biochem. J. 255, 541-545]. The tube-worm haemoglobins are believed to have a function of transporting sulphide (H2S) to internal bacterial symbionts, as well as of facilitating O2 transport [Arp & Childress (1983) Science 219, 295-297]. We have determined the complete amino acid sequence of Lamellibrachia chain AIII by automated or manual Edman sequencing. The chain is composed of 144 amino acid residues, has three cysteine residues at positions 3, 74 and 133, and has a molecular mass of 16,620 Da, including a haem group. The sequence showed significant homology (30-50% identity) with those of haem-containing chains of annelid giant haemoglobins. Two of the three cysteine residues are located at the positions where an intrachain disulphide bridge is formed in all annelid chains, but the remaining one (Cys-74) was located at a unique position, compared with annelid chains. Since the chain AIII was shown to have a reactive thiol group in the intact 3000 kDa molecule by preliminary experiments, the cysteine residue at position 74 appears to be one of the most probable candidates for the sulphide-binding sites. A phylogenetic tree was constructed from nine chains of annelid giant haemoglobins and one chain of vestimentiferan tube-worm haemoglobin now determined. The tree clearly showed that Lamellibrachia chain AIII belongs to the family of strain A of annelid giant haemoglobins, and that the two classes of Annelida, polychaete and oligochaete, and the vestimentiferan tube worm diverged at almost the same time. H.p.l.c. patterns of peptides (Figs. 4-7), amino acid compositions of peptides (Table 2) and amino acid sequences of intact protein and peptides (Table 3) have been deposited as Supplementary Publication SUP 50154 (13 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.     

A calorimetric study of the CO Bohr effect of monomeric haemoglobins.

A calorimetric study has been made of the heats of CO reaction with the monomeric haemoglobins of Chironomus thummi thummi III and IV as a function of pH. The number of Bohr protons released at pH 7.1 was determined from heats of reaction in different buffers as 0.19 and 0.31 mol H+/mol CO for haemoglobin III and IV respectively. The heat of the Bohr ionization process was found to be 6 and 8 kcal/mol H+ (25 and 34 kJ/mol) for the haemoglobins III and IV. These values are consistent with values found for histidine groups. A pH-independent part of the reaction enthalpy was determined as - 19.7 kcal/mol CO (-82.4 kJ/mol). The same reaction with myoglobin is less exothermic. From the combination of deltaG0 and deltaH0 values TdeltaS0 values have been calculated. It was found for both haemoglobins that the entropy of reaction is greater by 2 cal K-1 mol-1 (8.4 JK-1 mol-1) at pH 9.5 as compared to pH 6.0.     

A comparative approach to protein-and ligand-dependence of the Root effect for fish haemoglobins

Ligand-binding equilibria, kinetics and (13)C n.m.r. spectra of bound (13)CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the (13)CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O(2) strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the alpha and beta chains in the tetramer were obtained for both haemoglobins from (13)CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.     

Evolution of the haem-haem interaction in vertebrate haemoglobins - a hypothesis

A hypothesis about the evolution of the haem-haem interaction in haemoglobins has been formulated on the basis of available functional and structural data. It emerges that this cooperative muchanism is not necessarily due solely to the higher levels of protomer aggregation, because it occurs only in haemoglobins having the distal histidine. It is thus proposed that this amino acid residue might have had a significant role for the development of low-oxygen-affinity haemoglobins in vertebrates.     

Structural comparison of the haemoglobin components of the armoured catfish Pterygoplichthys pardalis. Evolutionary considerations.

Amino acid analyses and peptide mapping were performed for the four main haemoglobins from the armoured catfish Pterygoplichthys pardalis; component I, which is functionally distinct from the others, is structurally unique, whereas components II, III and IV, functionally indistinguishable, are closely related in structure. Compositional difference indices are calculated for the four components and for the two major haemoglobins from the trout Salmo irideus, and the results are discussed in terms of structural relationships and evolutionary history of fish haemoglobins.     

Functionally distinct haemoglobins of the cryopelagic Antarctic teleost Pagothenia borchgrevinki

Pagothenia borchgrevinki , has a higher haemoglobin concentration than other Antarctic notothenioids and the high oxygen capacity may correlate with the relatively active mode of life of this fish. The fish has five haemoglobins (Hb C, Hb 0, Hb 1, Hb 2 and Hb 3) with Hb 1 accounting for 70–80% of the total, and Hb C being present in trace amounts. Hb 1 and Hb 2 are functionally similar in terms of Bohr and Root effects. Hb 3 has a weaker Bohr effect than Hb 1 and Hb 2, and the Root effect is similar to that of Hb 1. Hb 0 has a strong Bohr effect and the Root effect is enhanced to a larger extent by the physiological effectors chlorides and phosphates than that of the other components with the exception of Hb C. The heats of oxygenation are lower than those of temperate fish haemoglobins. Temperature variations may have a different effect on the functional properties of each haemoglobin, and chloride and phosphates may play an important role in the conformational change between the oxy and deoxy structures. The complete amino acid sequences of Hb 1 and Hb 0, as well as partial N-terminal or internal sequences of the other haemoglobins, have been established. The high multiplicity of functionally distinct haemoglobins indicates that P. borchgrevinki , has a specialized haemoglobin system.     

Biochemische und cytogenetische Untersuchungen zur Natur des Hämoglobin-Polymorphismus bei Chironomus tentans und Chironomus pallidivittatus

The haemoglobin of chironomids is dissolved in the body fluid of the larvae and can be separated electrophoretically in Chironomus tentans into 10, and in C. pallidivittatus into 8, different bands. The molecular weight determined under the electrophoretic conditions was 15,000±1,000 for each haemoglobin band. This means that each haemoglobin band represented a single protein chain. In each species 7 haemoglobins could be characterised as species specific according to their different electrophoretic mobilities, developmental characteristics and the fact that one haemoglobin could be correlated genetically with a specific chromosome inversion. The inheritance of all these species specific haemoglobins was found to be co-dominant. With cytogenetic methods it was possible to define the loci of the species specific haemoglobin genes as being restricted to certain parts of chromosome 3. This finding suggests gene duplication as the most likely mecanism of the evolution of haemoglobins in Chironomus.     

A comparative study of haemoglobins from the Artiodactyla by isoelectric focusing.

1. Isoelectric focusing on 7% acrylamide gels in 2% ampholyte of pH range 6-8 was used to fractionate the haemoglobins of 81 animals, representing 30 species from 4 families of the Order Artiodactyla. 2. Isoelectric points of the major haemoglobins were determined and the relative distribution of minor haemoglobins compared. 3. Marked haemoglobin heterogeneity was observed, with polymorphic and multiple haemoglobins. No one haemoglobin was common to all species.     

Two Transitions of Haemoglobin Expression in Xenopus: from Embryonic to Larval and from Larval to Adult

Abstract. Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins ( X. borealis ) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.     

Recombination of embryonic haemoglobin chains during the development of the chick.

Embryonic differentiation is at present interpreted as the expression of variable gene activity. It is commonly thought that derepression of operator gene groups is the main cause of progress during development. However it is equally possible that gene repression plays a role in the appearance of new phenotypic characteristics. This paper illustrates such a possibility. It is known that in chickens embryonic haemoglobins exist which are replaced by other haemoglobins at about the sixth day of incubation. Analyses of globin chain composition of these haemoglobins by chromatography and urea/starch gel electrophoresis as well as TLC-fingerprinting and amino acid analyses of the individual globin chains showed that the haemoglobin switch was not associated with appearance of new globin chains but rather with disappearance of a number of embryonic chains. Moreover the relative proportion of the various chains changed at that time. From these findings we conclude that new haemoglobins arise from a recombination ('hybridization in vivo') of those globin chains which remain after the repression of a gene coding for embryonic chains.     

Two transitions of haemoglobin expression in Xenopus: from embryonic to larval and from larval to adult

Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins (X. borealis) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.     

Modulation of nitric oxide bioactivity by plant haemoglobins

Nitric oxide (NO) is a highly reactive signalling molecule that has numerous targets in plants. Both enzymatic and non-enzymatic synthesis of NO has been detected in several plant species, and NO functions have been characterized during diverse physiological processes such as plant growth, development, and resistance to biotic and abiotic stresses. This wide variety of effects reflects the basic signalling mechanisms that are utilized by virtually all mammalian and plant cells and suggests the necessity of detoxification mechanisms to control the level and functions of NO. During the last two years an increasing number of reports have implicated non-symbiotic haemoglobins as the key enzymatic system for NO scavenging in plants, indicating that the primordial function of haemoglobins may well be to protect against nitrosative stress and to modulate NO signalling functions. The biological relevance of plant haemoglobins during specific conditions of plant growth and stress, and the existence of further enzymatic and non-enzymatic NO scavenging systems, suggest the existence of precise NO modulation mechanisms in plants, as observed for different NO sources.     

Acid Bohr effect of a monomeric haemoglobin from Dicrocoelium dendriticum. Mechanism of the allosteric conformation transition

The dioxygen affinity of Dicrocoelium dendriticum haemoglobin was determined as a function of pH with a thin-layer diffusion technique. From the oxygen dissociation and association curves Hill coefficients h equal 1 were obtained throughout. Ultracentrifugation studies prove this haemoglobin to be monomeric irrespective of pH and ligation state. Thus, Dicrocoelium haemoglobin is a non-cooperative monomer. It has the highest O2 affinity so far known for any monomeric haemoglobin: its half-saturation pressure, p50 value, ranges at 25 degrees C from 0.016 mm Hg to 0.15 mm Hg (2.13-20.0 Pa) dependent on pH. Dicrocoelium haemoglobin shows an acid Bohr effect only and as such it constitutes a new class of haemoglobins. Its log p50 versus pH plot (Bohr effect curve) is characterized by a large amplitude, delta log p50 = 0.96, and an inflection point (Bohr effect pK) at pH 5.0. A model for the acid Bohr effect of D. dendriticum haemoglobin is proposed. By generalization, both the alkaline and the acid Bohr effect in various monomeric haemoglobins may arise from a single Bohr group complex (salt bridge).     

Nonsymbiotic haemoglobins in plants.

General aspects regarding the presence of nonsymbiotic haemoglobin in plants are presented with the emphasis on those related to its function. As it becomes apparent that the nonsymbiotic haemoglobins are widespread across the plant kingdom and that they represent a more primitive and evolutionary older form of the plant globin genes, the question of their function becomes more attractive. While the physiological functions of the symbiotic haemoglobins in plants are well understood, almost nothing is known about their nonsymbiotic predecessors. Therefore, the known and hypothetical functions of haemoglobins in various systems are described along with information concerning properties and the regulation of expression of the nonsymbiotic haemoglobins. Interestingly, a number of nonsymbiotic haemoglobins have been shown to be hypoxia-inducible. The spatial and temporal pattern of this induction in barley may suggest that it is an integral part of the plants response to limiting oxygen stress.     

Ontogenetic changes in haemoglobins in roach, Rutilus rutilus (L.) and rudd, Scardinius erythrophthalmus (L.)

The haemoglobins and globins of the roach, Rutilus rutilus (L.) and the rudd, Scardinius erythrophthalmus (L.) have been studied by starch gel electrophoresis. At all stages of development studied, the roach and rudd haemoglobins give a similar electrophoretic pattern, the young fish of about 2 cm standard length possessing three separate haemoglobins, of which two bands only persist in the adult. The evolutionary significance of multiple haemoglobins in fishes is briefly discussed.