JXB Advance Access originally published online on January 6, 2009
Journal of Experimental Botany 2009 60(2):409-422; doi:10.1093/jxb/ern320
REVIEW-ARTICLE |
Intrinsic non-symbiotic and truncated haemoglobins and heterologous Vitreoscilla haemoglobin expression in plants
1Department of Biology, PO Box 3000, 90014 University of Oulu, Finland
2Institute of Microbiology, ETH-Zürich, CH-8093 Zürich, Switzerland
* To whom correspondence should be addressed. E-mail: Soile.Jokipii-Lukkari{at}oulu.fi
Received 28 August 2008; Accepted 18 November 2008
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Abstract |
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 Top Abstract IntroductionStructural comparison of plant...Diverse functions of plant...Biological background and...Future challenges of plant...References |
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To date, haemoglobins (Hbs) have been shown to exist in all kingdoms of life. The least studied and understood groups are plant non-symbiotic haemoglobins (nsHbs) and the recently found plant truncated Hbs (trHbs). From a biotechnological point of view, the best characterized and almost exclusively applied Hb is the bacterial Vitreoscilla haemoglobin (VHb). In this review, the present state of knowledge of structural features and ligand binding kinetics of plant nsHbs and trHbs and their proposed roles as oxygen carriers, oxygen sensors, and for oxygen storage, in nitric oxide (NO) detoxification, and in peroxidase activity are described. Furthermore, in order to predict the functioning of plant Hbs, their characteristics will be compared with those of the better known bacterial globins. In this context, the effects of heterologous applications of VHb on plants are reviewed. Finally, the challenging future of plant Hb research is discussed.
Key words: Bacterial globins, non-symbiotic, plant haemoglobin, truncated, Vitreoscilla haemoglobin
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Introduction |
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TopAbstractIntroductionStructural comparison of plant...Diverse functions of plant...Biological background and...Future challenges of plant...References |
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At present, ubiquitous haemoglobins (Hbs) have been shown to exist in all kingdoms of life (Vinogradov et al., 2006). Putative globins, also including the group of globin-containing sensor proteins, have been identified in 426 bacterial, 32 Archaeal, and 67 eukaryote genomes (Vinogradov et al., 2007). However, "true" globins have been characterized only in 264 bacterial, eight Archaeal and 54 eukaryote genomes (Vinogradov et al., 2007). The bacterial globins are generally divided into three types of Hb proteins: truncated haemoglobins (trHbs), haemoglobins (Hbs), and flavohaemoglobins (flavoHbs). The best characterized and almost exclusively applied Hb in microbe and plant biotechnology is Vitreoscilla haemoglobin (VHb) (Frey and Kallio, 2003, 2005; Kallio et al., 2008).
In plants, upon binding of CO and O2, haemoproteins with absorption spectra typical of Hbs had already been discovered in soybean root nodules by Kubo in 1939. These globins are today termed as symbiotic or leghaemoglobins (sHbs), and their role in facilitating oxygen diffusion to nitrogen-fixing bacteria in the nodules of plants capable of symbiotic nitrogen fixation is also well described (Appleby, 1984; Appleby et al., 1988). Plant non-symbiotic haemoglobins (nsHbs) were first found in the roots of the tropical trees Parasponia andersonii and Trema tomentosa (Bogusz et al., 1988). Since these findings, expression of nsHbs has been reported in several plant species, especially in crop plants, as recently reviewed by Hoy and Hargrove (2008). The most recently found and still poorly characterized group of plant Hbs are trHbs. Plant TrHb genes were first discovered by Watts et al. (2001), and even today the number of trHbs characterized in plants is low and their role remains obscure.
The aim of this review is to introduce the present state of knowledge of nsHbs and trHbs in plants. In order to predict the functioning of plant Hbs, their characteristics are compared with those of the better known bacterial globins. In this context, the effects of heterologous applications of VHb in plants are reviewed. Finally, the future challenges of plant Hb research are also discussed.
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Structural comparison of plant non-symbiotic and truncated haemoglobins and their bacterial counterparts |
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TopAbstractIntroductionStructural comparison of plant...Diverse functions of plant...Biological background and...Future challenges of plant...References |
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Plant non-symbiotic haemoglobins
In plants, the nsHbs were originally divided into class-1 (Hb1) and class-2 (Hb2) subgroups (Trevaskis et al., 1997; Hunt et al., 2001), the differentiation of which is based on phylogenetic analyses, gene expression patterns, and oxygen-binding properties of the encoded proteins. The first crystal structure of monocot class-1 nsHb was that of rice (riceHb1) (Hargrove et al., 2000). The crystal structure of riceHb1 displays a typical hexacoordination, with the distal histidine linked to the haem iron, a bend in the E-helix, absence of the D-helix, disorder in the CD-region and a conserved phenylalanine (Phe-B10) in the B-helix that interacts with the distal histidine (Hargrove et al., 2000; Smagghe et al., 2006). However, there are also species-dependent differences within the monocots. For instance, the tertiary stucutures of riceHb1 and barley Hb (barHb1) are different (Bykova et al., 2006; Hoy and Hargrove, 2008). In corn, the computational modelling predicted a structure similar to riceHb1 (Saenz-Rivera et al., 2004), but cornHb1 has more interhelical bonds than riceHb1, which suggests reduced flexibility of corn Hb1 and putative kinetic differences in ligand binding (Smagghe et al., 2006).
The structures of nsHbs in dicots are not well known. In tomato, nsHb1 reveals hexacoordination in the ferric form and a mixture of pentacoordination and hexacoordination in the ferrous form (Ioanitescu et al., 2005). In Arabidopsis, AtHb1 (class-1 Hb, also referred to as AtGLB1 or AHb1 in the literature) is present at a 40% fraction in a pentacoordinated state and the distal haem cavity is suggested to be connected to the exterior by a relatively open channel (Bruno et al., 2007). The structure and functioning of class-2 nsHbs have been more precisely described only in the case of Arabidopsis AtHb2 (also referred to as AtGLB2 or AHb2) (Trevaskis et al., 1997; Bruno et al., 2007). In AtHb2, unlike in AtHb1, protein dynamics have a major role in the ligand migration from the distal cavity to the solvent (Bruno et al., 2007).
Bacterial haemoglobins and flavohaemoglobins versus plant non-symbiotic haemoglobins
In bacteria, the Hbs and flavoHbs exhibit a classical 3-on-3 
-helical globin fold, and share a high degree of sequence homology and structural similarity in their globin domain, the size of which is 
150 residues. FlavoHbs, which have not been identified in plants, are chimeric proteins (400 residues). In addition to the N-terminal oxygen-binding Hb domain, they possess a flavin-carrying reductase domain of the ferredoxin-NADP+ reductase family, which is able to transfer electrons from NADPH via FAD to the haem group (Karplus et al., 1991; Frey and Kallio, 2003, 2005; Gardner, 2005).
VHb is a homodimeric pentacoordinate Hb, adopting the classical Hb fold, where the haem is embedded in a hydrophobic crevice formed by helices B, C, E, F, G, and H (Tarricone et al., 1997). No protein–haem interactions are detected at the distal site. The proximal site residues His-F8, Tyr-G5, and Glu-H23 form a hydrogen bond network which modulates the redox properties of the haem in a similar fashion to that observed for cytochrome c peroxidase (Goodin and McRee, 1993; Mukai et al., 2001). These residues are also present in flavoHbs (Ermler et al., 1995; Ilari et al., 2002), which possess a well established function in nitric oxide (NO) detoxification. This suggests a role different from oxygen binding and storage of bacterial globin proteins. Notably, Lys-F7, which has been implicated in the transfer of electrons from FAD to the haem iron in flavoHbs, is present in VHb but absent from other bacterial Hb proteins (Ermler et al., 1995; Tarricone et al., 1997; Ilari et al., 2002).
Similar to bacterial Hbs and flavoHbs, plant nsHbs also exhibit a classical 3-on-3 -helical globin fold. In contrast to their pentacoordinated bacterial homologues and plant symbiotic Hbs, plant nsHbs are mostly hexacoordinated, as also observed for neuroglobin in humans (Trent et al., 2001) and cyanoglobin in cyanobacteria (Hvitved et al., 2001).
Sequence comparison of bacterial globins, represented by VHb and Escherichia coli flavoHb (Hmp), and of plant nsHb reveals that the residues Tyr-G5 and Glu-H23 implicated in the activation of the haem-bound ligand are missing (Fig. 1). Three similarly interesting observations have been made. First, Tyr-B10, which has been suggested to aid in the stabilization of the haem ligand in bacterial globin proteins, is only present in trHb and symbiotic leghaemoglobin but exchanged for phenylalanine in plant nsHbs (Smagghe et al., 2006). Secondly, the distal His-E7 present in hexacoordinate Hbs is replaced by glutamine in bacterial globins. The replacement of Tyr-B10 by phenylalanine strongly increased the O2 dissociation constant of the Hmp of E. coli (Gardner et al., 2000). Thirdly, Lys-F7, which is involved in the transfer of electrons from the FADH present in the reductase domain of flavoHb or from a non-linked reductase in the case of VHb, is also potentially missing. This might indicate that the mechanism for reducing the oxidized haem iron to the ferrous form involves a protein which is not structurally related to ferredoxin-NADP+ type reductase (Karplus et al., 1991). The importance of the reductase domain for detoxification of NO has been nicely demonstrated for VHb and very recently also for hexacoordinated Hbs (Frey et al., 2002; Smagghe et al., 2008).
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Truncated haemoglobins in plants and bacteria
TrHb is a rather new class of small myoglobin-like (2-on-2) proteins, which are widely distributed and can be identified in bacteria, unicellular eukaryotes, and plants. In bacteria, their primary structure is usually
20–40 residues shorter than the classical (non-)vertebrate Hb proteins and myoglobin, but the length can vary significantly, as seen in mycobacteria (121–215 residues) (Ascenzi et al., 2007). In contrast, the identified plant TrHb genes are longer than genes encoding 3-on-3 Hbs (Vinogradov et al., 2006; Jokipii et al., 2008). This new globin family can be divided, based on sequence clustering, into three groups in bacteria: I (trHbN), II (trHbO), and III (trHbP) (Wittenberg et al., 2002; Vinogradov et al., 2007). The crystallographic analyses have shown that all three trHb groups adopt a 2-on-2 -helical fold in which helices B, E, G, and H surround the haem (Nardini et al., 2007). The phylogenetic analysis conducted by Vuletich and Lecomte (2006) suggests that trHbO is the ancestral gene and groups I and III are the result of various replacement events. Structural analyses of plant trHbs are still scarce. The group I trHb of Chlamydomonas eugametos (CeTrHb) has an apolar tunnel that connects the protein surface to the distal haem pocket with xenon-binding sites and modulation of carbonmonoxy derivatives along the tunnel, which indicate putative ligand binding in the tunnel (Samuni et al., 2003; Milani et al., 2004a, b; Hoy and Hargrove, 2008). In Arabidopsis, the group II trHb (AtTrHb and also called AtGLB3 or AHb3) adopts a pentacoordinate state with a transient hexacoordinate state (Watts et al., 2001).
Ligand binding kinetics
Similar to other globin proteins, Hbs can bind a number of different ligands, including O2, CO, NO, and CN. Ligand binding kinetics that cover examples of plant Hbs, bacterial Hbs, and flavoHbs are summarized in Table 1. Intriguingly, bacterial flavoHbs possess a high autoxidation rate under aerobic conditions, with a rapid consumption of NADH (Farrés et al., 2005). Despite their high sequence similarity, the ligand-binding properties of flavoHbs and Hbs differ considerably, but the ligand-binding constants of a specific globin are independent of the presence of a reductase (Farrés et al., 2007). O2 and CO affinity constants and rates of NO binding to the ferric form of bacterial Hbs are roughly one order of magnitude higher than the respective values from flavoHbs and plant nsHbs, resembling more the oxygen affinities of sHbs. The observed biphasic behaviour for ligand binding of bacterial pentacoordinate globins can be attributed to the displacement of a lipid ligand from the haem pocket (Bonamore et al., 2003). NsHbs possess similar kon values for O2 relative to bacterial globins but generally lower koff, yielding higher affinity constants (Table 1). NO binding kinetics for ferrous plant nsHbs have not been determined; kon of ferric riceHb is five orders of magnitude lower than the corresponding values for Hbs and flavoHbs (Gardner et al., 2000; Farrés et al., 2005, 2007; Smagghe et al., 2008).
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Diverse functions of plant non-symbiotic and truncated haemoglobins compared with bacterial haemoglobins and flavoglobins |
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TopAbstractIntroductionStructural comparison of plant...Diverse functions of plant...Biological background and...Future challenges of plant...References |
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Physiological functions of plant non-symbiotic haemoglobins
Since their discovery, several important biochemical and physiological roles have been suggested for plant Hbs, including functioning as oxygen carriers, in oxygen storage, and as oxygen sensors, ligand transport, scavenging, sensing, detoxification, and electron transfer (Arredondo-Peter et al., 1998). The recent silencing and knockout studies on Arabidopsis thaliana have emphasized the vital role of nsHbs during plant development by showing that at least one functional nsHb gene is essential for survival of young seedlings (Hebelstrup et al., 2006).
To date, nsHb1 genes have been reported to be up-regulated by hypoxic, osmotic, and high salt stresses (Trevaskis et al., 1997; Lira-Ruan et al., 2001; Zhao et al., 2008), treatments with nitrate, nitrite, NO, salicylic acid, methyl jasmonic acid, ethylene, and H2O2 (Wang et al., 2000; Sakamoto et al., 2004; Ohwaki et al., 2005; Sasakura et al., 2006; Qu et al., 2006), deficiency of phosphorus, potassium, and iron (Wang et al., 2003), as well as dual culture with specific microsymbionts (Shimoda et al., 2005; Jokipii et al., 2008), whereas nsHb2s are known to be inducible by cold (Trevaskis et al., 1997) or cytokinins (Hunt et al., 2001). The characterization of Oryza sativa transcription factors regulating the expression of nsHb genes has indicated that cytokinin-induced activation of OsNSHb2 may be mediated by ARR1 (Arapidopsis Response Regulator 1) containing a Myb-like DNA-binding domain (Ross et al., 2004), while nsHb1 expression in tissues under hypoxic stress may depend on an incomplete transcription factor, Mybleu, unable to bind DNA (Mattana et al., 2007). Despite differential regulation, recent studies suggest that the functions of nsHb1s and nsHb2s also overlap (Ross et al., 2004; Hebelstrup et al., 2006). Moreover, recent database searches suggest that nsHb2 might be limited to specific plant species or genera (Jokipii et al., 2008).
Carriage, storage, or sensing oxygen
Historically, bacterial Hb proteins were considered to function as oxygen scavenging and delivering proteins, but their exact physiological function is still not known. After finding Hbs from non-nodulating plants (Landsmann et al., 1986), comparisons with globins of other organisms led to a hypothesis that plant nsHbs could possibly also function as general oxygen carriers, oxygen sensors, and for oxygen storage. However, nsHbs, especially Hb1s, have unusually high affinity for oxygen and they release oxygen slowly (Table 1), which are characteristics that later excluded these speculations on the function of nsHbs in oxygen delivery (Hill, 1998).
Peroxidase activity
Recently, VHb has been shown to possess peroxidase activity in vitro, reaching similar activity to horseradish peroxidase for 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) and ferrocene carboxylic acid, but acting poorly on phenolic compounds such as dopamine (Kvist et al., 2007). Interestingly, E. coli Hmp has alkylhydroperoxide reductase activity. Since the X-ray structures revealed the presence of lipids in the neighbourhood of the active site, there is speculation that this activity could help in the repair of oxidized membranes (Bonamore et al., 2003). The in vivo significance of this activity is not yet known. In plants, peroxidase activity has so far been shown in all three Arabidopsis Hbs, AtHb1, AtHb2, and AtHB3, representing nsHbs of class-1 and class-2 as well as trHb, respectively (Sakamoto et al., 2004). All three purified AtHb proteins, resolved by native PAGE, co-localized with peroxidase activity, but the signal generated by AtHb3 was considerably weaker relative to that of AtHb1 or AtHb2. In the activity staining, the oxidation of the chromogenic reagent was dependent on the presence of both H2O2 and the recombinant protein, and it was inhibited by KCN, thus revealing intrinsic peroxidase-like activity (Sakamoto et al., 2004).
NO detoxification
One of the biochemical properties suggested for VHb and more generally for bacterial globins as well as for plant Hb1 proteins is the neutralization of reactive nitrogen species (RNS) (Frey et al., 2002; Ouellet et al., 2002; Frey and Kallio, 2003, 2005; Igamberdiev and Hill, 2004; Gardner, 2005). FlavoHbs detoxify NO into nitrate under aerobic conditions and under consumption of NADH in a catalytic reaction called the nitric oxide dioxygenase (NOD) reaction (Gardner et al., 2000). Under physiological conditions with a limiting O2 concentration, flavoHb-bound NO reacts with O2 in a reaction termed the O2 denitrosylase reaction, which is consistent with a peroxidase-like function (Hausladen et al., 2001). In contrast to cells, cell-free extracts containing VHb were not able to degrade NO. This suggests that in vivo an endogenous reductase system has the capability to reduce the ferric VHb to the biochemically active ferrous form. However, upon preparation of cell-free extracts this reducing activity is lost (Frey et al., 2002). The enzymatic NOD activities of E. coli Hmp and Ralstonia eutropha flavoHb (FHP) are 94 s–1 (at 20 °C) and 7.4 s–1 (at 20 °C), respectively. It has been shown that a group I trHb in Mycobacterium bovis possesses efficient NO neutralizing activity and that M. tuberculosis oxygenated trHb is also able to oxidize NO into nitrate in stoichiometric amounts with a second-order rate constant of 745µM–1 s–1 (at 23°C) (Ouellet et al., 2002; Pathania et al., 2002).
The interplay between NO and Hb1s has usually been studied by treatments of plants with exogenous NO donors, e.g. sodium nitroprusside, or using NO-generating growth conditions. One of these conditions is hypoxic stress, which is known to increase the NO emission of plant cells (Dordas et al., 2003). This discovery has prompted intensive research on the possible functions of nsHbs under low oxygen tension. Sowa and co-workers (1998) were the first to observe that maize cells overexpressing barley Hb1 maintained ATP levels under hypoxic conditions more effectively than wild-type and Hb-negative cells. The NO concentration of the heterologous maize cells was later shown to have an inverse relationship with the level of Hb expression (Dordas et al., 2004). Moreover, partially purified recombinant alfalfa Hb1 was found to exhibit NO degradation activity and to rely on the use of NAD(P)H as a cofactor in vitro, but to lose this activity after the size exclusion chromatography step, indicating that Hb1 alone is not able to scavenge NO (Seregélyes et al., 2004). Based on the information gathered, Igamberdiev and Hill (2004) postulated that NO is produced during hypoxia through nitrate reduction in order to maintain the energy status of the cells. According to the authors, barley Hb1 prevents the accumulation of NO by acting as a dioxygenase, and turnover of this reaction is maintained by a methaemoglobin (metHb) reductase. Indeed, Igamberdiev et al. (2006) have recently identified a monodehydroascorbate reductase that is able to facilitate NO-scavenging activity of barley metHb in the presence of ascorbate and NADH. Furthermore, Smagghe et al. (2008) have recently compared, with various methods, NO reactions of rice, human (neuroglobin and cytoglobin), and Synechocystis haemoglobin with those of horse heart myoglobin. In vitro, all hexacoordinated oxygenated Hbs were able to destroy NO rapidly at a rate equal to the re-reduction of the ferric Hb. However, only Synechocystis Hb was able to replace the NO detoxification capability of E. coli flavoHb in vivo. Thus, Smagghe et al. (2008) suggested that future Hb research could be focused on the identification of possible reductases co-operating with Hbs in their natural environments. In early studies on VHb purifiction from its native host, a metHb reductase was co-purified (Webster and Liu, 1974; Gonzales-Prevatt and Webster, 1980). However, the elucidation of its function has not been pursued in the light of NO chemistry. Additionally, an alternative way for modulation of the NO level by Hbs has also been demonstrated by Perazzolli et al. (2004). They showed that A. thaliana AtHb1 protein scavanged NO through formation of S-nitrosohaemoglobin under hypoxic stress.
NO, a double-edged sword
NO has emerged as a key signalling molecule based on its chemical properties. NO can freely penetrate the lipid bilayer and hence be transported within the cell. NO is quickly produced on demand via inducible enzymatic or non-enzymatic routes. Due to its free radical nature (one unpaired electron), NO has a short half-life (in the order of seconds) and can be removed easily when no longer needed (Lamattina et al., 2003; Neill et al., 2003). In plants, NO has been shown to be involved in multiple processes, such as the expression of defence genes, programmed cell death, stomatal closure, seed germination, and senescense (reviewed by Neill et al., 2003).
NO is a double-edged sword for plants: on the one hand, it is toxic to cells but, on the other hand, together with ROS, it contributes to triggering a hypersensitive response (HR) to prevent the propagation of biotrophic pathogens (Delledonne et al., 1998). Despite the clear effects of Hb1 proteins on the abundance of NO under oxygen-limited conditions, studies with Hb1-overexpressing or -underexpressing lines treated with pathogens have resulted in controversial findings. Seregélyes and co-workers (2003) showed that transgenic tobacco plants overexpressing an alfalfa Mhb1 gene had reduced necrosis after pathogen infection, with either Pseudomonas syringae or tobacco necrosis virus. Additionally, Qu et al. (2006) reported enhanced resistance against P. syringae in A. thaliana plants that were genetically modified with GhHb1 of cotton. On the other hand, Perazzolli et al. (2004) did not observe any changes in NO accumulation or HR in AtHb1-overexpressing Arabidopsis plants inoculated with P. syringae relative to wild-type plants. In order to defend themselves against invading pathogens, plants have to have mechanisms for recognizing the advantageous and the harmful NO. In mammals, this differentiation is based on NO concentration. Whereas constitutively expressed nitric oxide synthase (NOS) produces NO in response to physiological stimuli, the inducible form of NOS produces NO as a defence molecule in high amounts (Coleman, 2001). How this discrimination between these two functions is achieved in plant systems remains currently largely unknown.
Functioning of truncated haemoglobins in plants
Because trHbs have only recently been discovered in higher plants (Watts et al., 2001), the number of studies analysing their functional roles is very limited and no body of information similar to that for some bacterial globins is yet available for trHbs. The expression of plant TrHb genes has been shown to remain uninduced under hypoxia or upon treatment with phytohormones (Watts et al., 2001), but Vieweg and co-workers (2005) observed the up-regulation of certain TrHb genes of Medicago truncatula Gaertn. during symbiotic association in root nodules and in roots colonized by arbuscular mycorrhizal fungi. Moreover, Pawlowski et al. (2007) showed that expression of the TrHb gene of Datisca glomerata is induced prior to the onset of bacterial nitrogen fixation in actinorhizal nodules. It was found later that the up-regulation of TrHb genes is not restricted to endosymbionts but it was observed that the ectomycorrhizal fungi Leccinum populinum and Xerocomus subtomentosus, with or without emergence of symbiotic structures, also increased the expression of both PttHb1 (encoding class-1 nsHb) and PttTrHb (encoding trHb) genes in Populus tremulaxtremuloides (Jokipii et al., 2008). PttHb1 and PttTrHb had expression peaks 5 h and 2 d after inoculation, respectively, which suggests different functions for these genes during interaction with ectomycorrhizal fungi.
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Biological background and biotechnological applications of VHb in plants |
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TopAbstractIntroductionStructural comparison of plant...Diverse functions of plant...Biological background and...Future challenges of plant...References |
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One of the main concerns in microbial biotechnology was, and still is, to find an efficient and cheap way to deliver oxygen to aerobic microorganisms during large-scale production processes. Historically, due to its extraordinarily high koff rate for oxygen release and its inducibility by O2 deprivation, VHb was considered for a long period of time an oxygen-binding and delivering protein (Orii and Webster, 1986). The synthesis of VHb seemed an obvious natural genetic strategy to combat oxygen limitation and improve phenotypic properties of Vitreoscilla. Therefore, it was hypothesized that heterologous expression of VHb could be used to alleviate oxygen limitation, and enhance oxygen delivery and the performance of industrially important bioprocesses. Motivated by this hypothesis, the VHb was first expressed in E. coli and the results showed that VHb enabled cells to reach higher cell densities under poorly oxygenated conditions (Khosla and Bailey, 1988). These successful experiments paved the way for future applications, and VHb has been expressed in numerous biotechnologically relevant microorganisms, and also later in eukaryotic cells and plants. In all these cases, positive effects on either cellular physiology and metabolism, cell growth, or production of industrially important compounds have been reported (Kallio et al., 2001, 2008; Frey and Kallio, 2003, 2005). Re-examination of the kinetic constants for O2 binding and release revealed a koff for O2 which is similar to that of other bacterial Hb proteins (Giangiacommo et al., 2001; Farrés et al., 2007). However, experiments directly revealing the biochemical function of VHb under these microaerobic conditions are still missing.
In bacteria, the expression of VHb has usually been obtained using the original vhb promoter (Khosla and Bailey, 1988; Frey and Kallio, 2003, 2005; Kallio et al., 2008). The vhb promoter is not induced by nitrosative or oxidative stress, although the VHb is able to neutralize NO in E. coli (Frey et al., 2003). However, expression of vhb is induced by oxygen deprivation. Its function has been thoroughly studied under microaerobic conditions, where it has been shown to change the gene expression pattern, increase the carbon flux through glycolysis, and enhance respiratory activity, leading to improved ATP synthesis and lowering the steady-state NAD(P)+/NADPH ratio in E. coli (Kallio et al., 1994; Tsai et al., 1995, 1996; Frey et al., 2001, 2007) Although these effects were formerly attributed to the oxygen delivering role, it is now believed that VHb enhances respiratory activity by shielding NO-sensitive cytochromes (Kaur et al., 2002). In contrast to the expression of VHb in E. coli, the native hmp gene is induced by nitrosative stress conditions and its function is clearly implicated in protection from nitrosative stress (Poole et al., 1996; Anjum et al., 1998; Cruz-Ramos et al., 2002).
It has also been hypothesized that the biochemical properties of VHb are not always optimal for foreign host cells. Therefore, VHb mutants showing highly improved growth properties in E. coli were generated using error-prone PCR (Andersson et al., 2000; Kallio et al., 2001). Unfortunately, the consequences on the kinetc properties for O2 and NO binding have not been elucidated. In addition, the expression of an engineered VHb fusion protein, ‘FlavoVHb’, carrying the C-terminal reductase domain of the R. eutropha hmp gene, was able to enhance growth and improve energetic characters of E. coli significantly (Frey et al., 2000, 2001). In the case of the chimeric VHb reductase fusion, the improvements in growth under microaerobic conditions correlate with the potentiating of its NO detoxification activity in comparison with VHb (Frey et al., 2002) The overwhelming genomic information has also allowed the cloning of novel bacterial Hb genes, yielding E. coli strains with improved phenotypical characters upon their expression (Bollinger et al., 2001; Kallio et al., 2007). This clearly shows that there is room for new developments, and other Hbs, natural or engineered, should also be tested for their efficacy in prokaryotic and eukaryotic cells and in plants.
Heterologous expression of Vitreoscilla haemoglobin in plants
As stated above, VHb and its engineered derivatives have been expressed in various heterologous hosts and have been shown to improve growth, cellular metabolism, and productivity of interesting products, e.g. antibiotics and amino acids in microorganisms, as summarized in Frey and Kallio (2003, 2005) and Kallio et al. (2008).
Due to the numerous successful achievements obtained in microorganisms, it is not surprising that Hb technology has also been tested in plants, and VHb has been the prime choice to be expressed in planta (Table 2). The pioneering work of Holmberg et al. (1997) with VHb revealed important phenotypic and metabolic changes, e.g. faster germination, enhanced growth, and changes in activities of metabolic pathways in Nicotiana tabacum, attributing the effects to increased intracellular oxygen levels. The follow-up study conducted using tobacco plant cell cultures also confirmed that VHb expression could be used to enhance growth and to generate more plant material relative to controls (Farrés and Kallio, 2002). Frey et al. (2004) were not able to repeat the experimental results of Holmberg et al. (1997) in tobacco, discerning no effect of VHb expression on growth. However, they were able to show that suspension cultures derived from VHb-expressing plants were more resistant against nitrosative stress. Furthermore, an enzyme assay for the NO-sensitive tricarboxylic acid cycle enzyme aconitase revealed that VHb was able to protect the NO-labile Fe–S cluster of aconitase against RNS inactivation (Frey et al., 2004).
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VHb has also been expressed in rice (Cao et al., 2004), potato (Zhou et al., 2004), and cabbage (Li et al., 2005). In cabbage, VHb-positive seeds exhibited a faster early germination rate than the wild-type controls, and the transgenic plants also showed improved tolerance to prolonged submergence treatment (Li et al., 2005). In rice, vhb was co-transformed and co-expressed with the Agrobacterium tumefaciens tzs gene encoding zeatin-type cytokinins and the mutated aroA gene from Salmonella typhimurium that renders recombinant plants resistant to glyphosate treatment (Cao et al., 2004). When the rice plants expressed both vhb and tzs genes, the plants were able to display a significant increase in plant height and panicle length relative to the non-transformed controls. Zhou et al. (2004) transformed potato plants by using expression vectors that carry a cry3A–vhb expression cassette. The cry3A gene encodes the coleopteran-specific endotoxin of Bacillus thuringiensis (Donovan et al., 1988). Interestingly, the water logging tests demonstrated that cry3A–vhb-positive potato plants were able to exhibit higher tolerance against low oxygen stress (Zhou et al., 2004). However, the studies on vhb gene expression alone without co-transformation with herbicide and cytokinin genes or herbivore resistance genes might have improved the possibilities to evaluate VHb effects in rice and potato, respectively.
In VHb-expressing hybrid aspen (P. tremulaxtremuloides), no general growth-promoting effects were obtained (Häggman et al., 2003). VHb-expressing lines had higher relative volumes of mitochondria and showed significantly enhanced starch accumulation in chloroplasts. Under elevated UV-B illumination, some specific VHb lines had elevated levels of total flavonoids, and individual quercetin, kaempferol, and myricetin derivatives. In the experiments conducted using VHb-producing Populus alba L. lines, Zelasco et al. (2006) could reveal positive effects on growth in only one out of six transgenic lines. In contrast to the findings of Frey et al. (2004), no positive effects of VHb on the growth of suspension cultures under nitrosative stress conditions relative to wild-type controls were detected. VHb has also been expressed in barley and in a medicinal plant, Hyoscyamus muticus (Egyptian henbane), that produces a commercially important tropane alkaloid scopolamine (see Table 2 for references and a summary of key findings).
While VHb expression may have erratic effects on plant growth and tolerance against various stresses, the studies evaluating the global gene expression and secondary metabolism of genetically modified lines could lead to a deeper understanding of functioning of heterologous VHb protein. Wang and co-workers (2008) have recently studied in detail VHb-expressing A. thaliana lines that exhibited an improved germination rate and tolerance against waterlogging, as well as nitrosative and photo-oxidative stresses. It was revealed that VHb expression significantly increased the abundance of vitamin C and phytosterol in leaf samples. When the expression of Arabidopsis endogenous genes involved in oxygen metabolism and antioxidant biosynthesis was analysed, the genes encoding glutathione reductase, glutathione synthase, ascorbate peroxidase, GDP-mannose pyrophosphorylase, and GDP-mannose showed up-regulation in comparison with control plants.
The improved starch production and enhanced growth could indicate that VHb-expressing lines have an improved energy household (Holmberg et al., 1997; Farrés and Kallio, 2002; Häggman et al., 2003). Such positive changes in energy status have also been reported when barley Hb (barHb) has been expressed constitutively in maize cells under limited oxygen availability. Under such conditions, the ATP levels were not affected in plants overexpressing barHb but were reduced by 27% in wild-type and 61% in Hb-negative cells, respectively (Sowa et al., 1998). Therefore, it has been hypothesized that nsHbs are needed in order to maintain the energy status, possibly by enhancing the substrate-level phosphorylation and concomitant NAD(P)H oxidation under diminished oxygen concentrations. This assumption is also supported by the findings of Igamberdiev et al. (2004), who reported lower NAD(P)H/NAD(P)+ ratios in alfalfa root cultures expressing barHb. The VHb-positive barley plants did not react to oxygen deficiency by increasing the alcohol dehydrogenase (ADH) activity in the roots relative to the control plants (Wilhelmson et al., 2007). Such an observation could mirror the ability of VHb to substitute ADH for recycling of NADH and to maintain glycolysis, and would require a high autoxidation rate of the globin protein and an efficient reductase system. Indeed, VHb and other bacterial globin proteins display a high autooxidation rate (Farrés et al., 2005, 2007). However, the endogenous plant reductase system capable of reducing ferric VHb remains elusive. Overall, these results parallel the early findings of the effects of VHb expression on microaerobic metabolism in E. coli (Kallio et al., 1994; Tsai et al., 1995).
Although, heterologous expression of VHb has been shown to generate important metabolic effects, it is still the only bacterial Hb which has been expressed in plants. Therefore, it is surprising that neither engineered nor new Hbs, showing improved properties in bacteria, have been tested in plants yet.
Interplay betgween endogenous haemoglobins and VHb
Due to the similarities between the suggested roles of VHb and plant endogenous Hbs in the NO reactions (Dordas et al., 2003, 2004; Frey et al., 2004), the expression of the genes has also been examined simultaneously during root growth (Wilhelmson et al., 2007; Jokipii et al., 2008), the developmental stage that is known to be regulated by NO (Pagnussat et al., 2002; Correa-Aragunde et al., 2004). The western analyses in barley indicated that the abundance of Hb1 was slightly lower in normoxic roots of VHb-expressing lines than in control plants, but the difference was not observed in anoxic root tissues (Wilhelmson et al., 2007). In hybrid aspen, the expression of endogenous PttHb1 and PttTrHb genes was studied by real-time RT-PCR in the roots of control and VHb-expressing plants that were cultivated with and without root growth-increasing ectomycorrhizal fungi (Jokipii et al., 2008). The results indicated that PttHb1 and PttTrHb genes were up-regulated in the roots of non-transgenic control lines during the dual culture with ectomycorrhizal fungi. Surprisingly, the fungi were not able to enhance the expression of endogenous Hb genes in the VHb lines, which suggests that endogenous Hbs may relate to early growth responses caused by fungi and that VHb may compensate the function of endogenous Hbs (Jokipii et al., 2008).
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Future challenges of plant haemoglobin research |
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TopAbstractIntroductionStructural comparison of plant...Diverse functions of plant...Biological background and...Future challenges of plant...References |
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The fascinating world of haemoproteins covers all kingdoms of life and interests of researchers in different disciplines, from medicine to plant sciences. Research on plant hexacoordinated nsHbs, and especially trHbs, is still in its infancy, although a lot of information has been accumulated during recent years. The new sequencing techniques will foster the sequencing of plant genomes and widen the repertoire of Hb sequences from the present model plants (such as A. thaliana, M. truncatula, O. sativa, Zea mays, and Hordeum vulgare) to more genera and species. This will certainly provide new information about the structure and biochemical properties of Hbs but will also verify the validity of the present grouping into class-1 and class-2 nsHbs. Such a clarification would be important because recent database searches already suggest that class-2 Hbs might be limited to specific plant species or genera (Jokipii et al., 2008). Additionally, it will be interesting to know if corresponding groups of trHbs that have been encountered in bacteria can also be identified in plants.
The hexacoordinated Hbs are, generally, known for their high cross-species sequence identity, indicating proteins with important physiological roles. However, although there are several hypotheses on their roles, their physiological functions still remain obscure. Thus, more biochemical, biophysical, structural, and protein-level research is needed to enable their physiological importance to be understood. For instance, the recent silencing and knockout studies on A. thaliana have emphasized the important role of nsHbs during plant development (Hebelstrup et al., 2006), but their physiological role is still elusive.
So far, very few scientific data have been gathered from plant trHbs and, for instance, structural and other physiological features have to be characterized thoroughly. Although there is convincing evidence that plant class-1 nsHbs are able to detoxify NO, some essential questions about their functioning and properties still need to be resolved. In plant cells, NO has been localized to mitochondria, cytosol, peroxisomes, and apoplasts (Blokhina and Fagerstedt, 2006), whereas nsHb1 proteins are apparently distributed throughout the nucleus and cytosol (Seregélyes et al., 2000; Hebelstrup et al., 2008) but are absent from mitochondria and peroxisomes (Hebelstrup et al., 2007). Inevitably, this raises a question about the role of nuclear Hb1, which in alfalfa cells represents the majority of the Hb1 proteins (Seregélyes et al., 2000). Moreover, it has recently been reported that NO dioxygenase activity in hexacoordinated haemoglobins is ubiquitous in vitro but limited by reduction in vivo. This clearly indicates that cognate reductases for each Hb within their natural environments have to be identified and analysed (Smagghe et al., 2008).
Heterologous VHb expression has been shown to improve growth, cellular metabolism, and productivity of interesting products (Table 2). The achieved results in plants parallel well the early findings of the effects of heterologous VHb expression on microaerobic metabolism in bacteria (Frey and Kallio, 2003). The beneficial role of VHb in plant biotechnology has thus been emphasized, although some controversial results have also been reported (Table 2). It seems obvious that heterologous VHb expression, not excluding other potentially interesting bacterial Hbs or engineered variants (Andersson et al., 2000; Frey et al., 2000; Bollinger et al., 2001; Kallio et al., 2001, 2007, 2008), should be analysed on a case-by-case basis. Such experiments may reveal if VHb can be used to improve the energy status of specific plant species, potentially leading to economically important applications in the bioenergy field. On the other hand, starch accumulation can potentially lead to improved growth or carbon sequestration to secondary metabolites under suboptimal environmental conditions (e.g. under flooding in nature). New and fast screening methods and reliable high-throughput cultivation techniques are also needed to evaluate the biotechnologically important characters of Hbs originating from microorganisms and plants.
To conclude, many questions still remain open in the fascinating world of haemoproteins, especially in the case of plant nsHbs and trHbs. We will certainly learn a lot from the bacterial counterparts that are at the moment better characterized and known, but a great deal of research needs to be carried out concerning plants. Future research on plant nsHbs and trHbs will be challenging.
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Acknowledgements |
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The authors are grateful to the Academy of Finland for financial support (grant no. 123826 to HH).
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Abbreviations |
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ADH, alcohol dehydrogenase; Hb, haemoglobin; HR, hypersensitive response; NO, nitric oxide; NOD, nitric oxide dioxygenase; NOS, nitric oxide synthase; nsHb, non-symbiotic haemoglobin; RNS, reactive nitrogen species; sHb, symbiotic haemoglobin; trHb, truncated haemoglobin.
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