JXB Advance Access originally published online on September 24, 2004
Journal of Experimental Botany 2004 55(408):2473-2482; doi:10.1093/jxb/erh272
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REVIEW ARTICLE |
Nitrate, NO and haemoglobin in plant adaptation to hypoxia: an alternative to classic fermentation pathways
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
* To whom correspondence should be addressed. Fax: +1 204 474 7528. E-mail: rob_hill{at}umanitoba.ca
Received 5 May 2004; Accepted 11 August 2004
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Abstract |
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 Top Abstract IntroductionNitrate uptake and anaerobiosisRegulation of nitrate reductases...Role of haemoglobinThe Hb/NO cycle and...ConclusionReferences |
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The role of nitrate reduction to produce nitric oxide (NO) and its subsequent oxidation by oxyhaemoglobin as a mechanism to maintain plant cell energetics during hypoxia is examined. Nitrate reduction in hypoxic conditions can be considered as an alternative respiratory pathway, with nitrate as an intermediate electron acceptor, contributing to the oxidation of NADH. NO, produced in the reaction, does not accumulate due to the induction of hypoxia-induced (class 1) haemoglobins. These haemoglobins remain in the oxyhaemoglobin form, even at oxygen tensions two orders of magnitude lower than necessary to saturate cytochrome c oxidase. They act, probably in conjunction with a flavoprotein, as NO dioxygenases converting NO back to nitrate, consuming NAD(P)H in the process. The overall system oxidizes 2.5 moles of NADH per one mole of nitrate recycled during the reaction, leading to the maintenance of redox and energy status during hypoxia and resulting in the reduced production of ethanol and lactic acid.
Key words: Glycolysis, haemoglobin, hypoxia, nitrate reductase, nitrite reductase, nitric oxide
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Introduction |
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TopAbstractIntroductionNitrate uptake and anaerobiosisRegulation of nitrate reductases...Role of haemoglobinThe Hb/NO cycle and...ConclusionReferences |
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ATP synthesis, required to maintain plant growth and viability in an aerobic environment, is achieved by the oxidation of carbon sources using oxygen as an electron acceptor. Under conditions that limit oxygen availability, complete substrate oxidation is restricted by the lack of an electron acceptor. Production of ethanol and lactic acid through well-known fermentation pathways is one mechanism that organisms use to provide glycolytic substrate oxidation and ATP synthesis, maintaining short-term cell viability under hypoxic conditions. There is evidence now of a second process operating that may be a critical factor for plant survival in hypoxic environments (Dordas et al., 2003a
, b, 2004; Igamberdiev et al., 2004b). The process (Fig. 1) involves a stress-induced (class 1) haemoglobin, with a high affinity for oxygen, and nitric oxide, produced via nitrite reduction. NADH oxidation is achieved in the reactions forming NO and its subsequent oxidation back to nitrate. The high affinity of the haemoglobin for oxygen facilitates the cyclic process, even at extremely low oxygen tensions. In this review, the experimental evidence that lends support to this hypothesis is summarized.
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Nitrate uptake and anaerobiosis |
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TopAbstractIntroductionNitrate uptake and anaerobiosisRegulation of nitrate reductases...Role of haemoglobinThe Hb/NO cycle and...ConclusionReferences |
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The switch that occurs from the aerobic tricarboxylic acid cycle to fermentation pathways, forming lactate and ethanol under hypoxic or anoxic conditions, is accompanied by a decline in pH due to ATP hydrolysis and lactate accumulation (the latter may be out of step with cytoplasmic acidification and may even not occur in some hypoxic tissues) (Saint-Ges et al., 1991
; Kennedy et al., 1992; Ratcliffe, 1995; Gout et al., 2001). The decline in pH slows the rate of lactate formation (Hanson and Jacobsen, 1984) and activates pyruvate decarboxylase, diverting glycolytic carbon flow to ethanol formation (Kennedy et al., 1992). Alanine, formed via the amination of pyruvate, is second only to ethanol as a product of glycolytic flux during hypoxia (Gibbs and Greenway, 2003). Unlike ethanol formation, the formation of alanine does not consume NADH, potentially leading to a decline in glycolytic flux due to the unavailability of an electron acceptor.
A portion of the requirement for an electron acceptor may be supplied via nitrate reduction, providing the 
required in the transamination reaction forming alanine. Evidence that 15N, supplied as exogenous 
appears in amino acids (Reggiani et al., 1993; Fan et al., 1997), the de novo synthesis of enzymes of the 
reduction pathway (Mattana et al., 1994), and the activation of nitrate reductase during hypoxia as a result of a decline in pH (Botrel and Kaiser, 1997) suggest that nitrate reduction plays an important role during hypoxia (Botrel et al., 1996). Alkalization of the medium observed during nitrate uptake (Steffen et al., 2001) may be linked to the prevention of acidification of the cytosol in hypoxic conditions.
Estimates of the end-products of nitrate reduction suggest that only 1.3–4% of NADH recycled during hypoxia is connected with the reduction of nitrate to (Gibbs and Greenway, 2003). The same authors argue that products, such as alanine, have not been detected in sufficient amounts to consider them as alternatives to the classic fermentation products in accounting for glycolytic flux. There are, however, experimental estimates of 30% NAD recycling, possibly as a result of nitrate reduction (calculated from the observed decrease in ethanol production in nitrate-fed cells) (Fan et al., 1997). In the very anoxia-tolerant Echinochloa phyllopogon, ethanol and lipid synthesis account for only 34% oxidation of NAD(P)H, while the rest can be linked to nitrate reduction (Fox et al., 1994). A lower rate of ethanol production in rice coleoptiles supplied with compared with those supplied with has been shown (Fan et al., 1997). Considering NO as a possible product of nitrite reduction from nitrate reductase (Fig. 1), it is likely that only a minor part of the nitrite formed during hypoxia will undergo reduction to ammonia (Figs 1, 2) since nitrite reductase is inhibited under these conditions (Botrel et al., 1996).
Electron microscopic studies of rice mitochondria during anaerobiosis clearly show a protective role of nitrate in maintaining membrane ultrastructure (Vartapetian and Polyakova, 1999; Vartapetian et al., 2003). These ultrastructural observations show the positive effect of nitrate reduction on the functionality of the anaerobic plant cell. It has been noted that ATP is maintained in potato cells at a higher level under hypoxia in nitrate as opposed to ammonium ion medium (Oberson et al., 1999; Rawyler et al., 2002). These findings suggest that, during inhibition of the mitochondrial electron transport chain by lack of oxygen, the presence of nitrate may have an influence, however indirect, on the functional state of mitochondria.
Alanine formation is strongly induced during hypoxia. There is no consumption of NADH in the process, while the 70% excess NADH due to alanine synthesis might have been recycled via nitrate reduction to ammonium (Gibbs and Greenway, 2003, assessed from Reggiani et al., 1995). Ammonium ion, from nitrate reduction, can be incorporated into glutamate either via the GS-GOGAT system consuming NADPH and ATP or via reverse glutamate dehydrogenase consuming only NAD(P)H, a more favourable reaction in hypoxic conditions (Gibbs and Greenway, 2003). A portion of the glutamate can be converted to 
-aminobutyric acid, which is suggested to be an important component for pH regulation (Crawford et al., 1994). The concentration of 2-oxoglutarate is maintained via a partial TCA cycle operating at elevated reduction levels in mitochondria (Igamberdiev and Gardeström, 2003) that are characteristic under hypoxic conditions. The decrease of 2-oxoglutarate dehydrogenase activity in anaerobically grown rice and Echinochloa while other TCA cycle enzymes were present at high levels (Fox and Kennedy, 1991) is in favour of 2-oxoglutarate accumulation. Figure 2 summarizes the metabolic pathways linked to nitrite reduction and the formation of ammonium ion. It should be noted that alanine appears as a product of nitrite reduction to ammonium ion and its formation strongly depends on the operation of nitrite reductase which is inhibited under hypoxia (Botrel et al., 1996).
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Regulation of nitrate reductases and NO formation |
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TopAbstractIntroductionNitrate uptake and anaerobiosisRegulation of nitrate reductases...Role of haemoglobinThe Hb/NO cycle and...ConclusionReferences |
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In roots, two distinct types of nitrate reductase are present, one located in the cytosol (cNR) and the other attached to the plasma membrane and facing the apoplast (PM-NR) (Stöhr and Ullrich, 1997
; Stöhr and Mäck, 2001). Estimates show that, in general, only one-third of root nitrate reduction can be attributed to cNR, while two-thirds may be due to PM-NR (Gojon et al., 1986).
There is a 2.5-fold activation of cNR during exposure of plant roots to hypoxia (Botrel and Kaiser, 1997), with nitrite reduction being suppressed at the nitrite reductase step (Botrel et al., 1996; Ferrari and Varner, 1971). The limitation of nitrite reduction is connected both with cellular acidification and with increased flux through nitrate reductase (Botrel and Kaiser, 1997; Botrel et al., 1996). The potential maximum activity of activated nitrate reductase, although lower than alcohol dehydrogenase, exceeds the rate of hypoxic ethanol formation by more than 3-fold (Botrel and Kaiser, 1997). In Arabidopsis root cultures, two nitrate reductase genes were induced under low-oxygen (5%) pressure. The NR1 gene showed moderate induction after 0.5–4 h of hypoxia and strong induction after 20 h. The NR2 gene was strongly activated in 2–4 h and even more after 20 h (Klok et al., 2002).
Nitrate reductase is inactivated by the interaction of the phosphorylated form of the enzyme with 14-3-3 proteins (Huber et al., 2002), while the enzyme is activated by dephosphorylation, catalysed by cNR phosphatase. Changes in cNR activity measured in vitro are not always associated with changes in nitrate reduction rates in vivo, suggesting that the cNR can be under strong substrate and/or cofactor limitation. The degradation and half-life of the cNR protein also appears to be affected by cNR phosphorylation and 14-3-3 binding, as cNR activation always correlates positively with its stability (Kaiser and Huber, 2001).
In aerated roots, cNR is highly phosphorylated and largely inactive. It is partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid) (Botrel and Kaiser, 1997). Anaerobic activation of NR is about 2.5-fold greater at acidic external pH than at slightly alkaline pH, although ATP levels decrease and AMP levels increase at pH 5 and at pH 8 to the same extent (Kaiser et al., 1999). Thus, rapid changes in the cNR-phosphorylation state in response to anaerobiosis are not directly triggered by the adenylate pool, but rather by cytosolic pH. Although some authors state that an increase in cNR activity does not prevent ethanol formation (Botrel and Kaiser, 1997), the nitrate reductase-lacking plants of tobacco produced substantially more ethanol and lactate during anaerobiosis (Stoimenova et al., 2003) indicating that cNR may have the effect of directing hypoxic metabolism away from lactate and ethanol formation.
Plasma membrane nitrate reductase (PM-NR) activity was initially demonstrated by Huffaker's group (Ward et al., 1988, 1989; Meyerhoff et al., 1994). It is present only in root tissue where it exceeds the activity of cNR, particularly during the night (Stöhr and Mäck, 2001). It can use both succinate and NADH, but succinate is the preferred electron donor. Taking into account succinate accumulation during hypoxia (Fan et al., 2003) and the possibility of fumarate reduction back to succinate by succinate dehydrogenase in co-operation with complex I under the accumulation of reduced ubiquinone (Cecchini, 2003), there is the possibility that the plasma membrane may have an important role in nitrate reduction during hypoxic conditions.
Plasma membrane-bound nitrite-NO reductase (Ni-NOR) is the likely enzyme that converts nitrite to NO rather than PM-NR. Ni-NOR faces the apoplast and has an activity sufficient to convert all of the nitrite formed by PM-NR to NO (Stöhr et al., 2001). Ni-NOR uses reduced cytochrome c for nitrite conversion to NO (Stöhr et al., 2001). Since participation of cytochrome c at the plasma membrane is unlikely, it is possible that the physiological electron donor for this reaction could be either another cytochrome or Hb, induced under hypoxic condition (Fig. 1). A haem protein oxidized during this reaction can be reduced by a protein possessing cytochrome reductase activity. The pH optimum of Ni-NOR is favourable for hypoxic conditions (pH 6.1), and it can utilize even low amounts of nitrite (Vmax is reached at a nitrite concentration of 100 µM) (Stöhr et al., 2001).
There is a possibility that plant Ni-NOR may perform a similar function to the respiratory nitrite reduction of micro-organisms (Stöhr and Ullrich, 2002) and mammalian mitochondria (Kozlov et al., 1999). It is known that NO is a potent inhibitor of the mitochondrial cytochrome c oxidase (complex IV) (Zottini et al., 2002). The alternative cyanide-resistant oxidase is NO-resistant, however, its participation in the hypoxic operation of mitochondria is unlikely because of its low affinity to oxygen (Huang et al., 2002).
Modulation of the redox state of the plasma membrane in hypoxia is indicated by the formation of hydrogen peroxide, detectable by electron microscopy, indicating the possible operation of an NADPH oxidase (Blokhina et al., 2001). Nitrate uptake can be regulated by plasmalemma redox activity (Steffen et al., 2001). The H+-ATPase of the plasma membrane and cytosolic nitrate reductase are both regulated by 14-3-3 proteins (Finnie et al., 1999). Although it is not clear how nitrate uptake, NO formation, and scavenging at the site of the plasma membrane can be related to the maintenance of ATP and NADH levels during hypoxia, there may be similar systems operating in plants to those that have been suggested in micro-organisms (Jormakka et al., 2003). Protonmotive force generation via a redox loop mechanism on the plasma membrane may include NAD(P)H oxidases linked putatively to PM-NR and/or Ni-NOR via cytochrome b5 and phylloquinone found in plasmalemma preparations (Bridge et al., 2000). Phylloquinone function may be similar to the function of mitochondrial ubiquinone (Lochner et al., 2003). The presence of multiple redox proteins in plasma membrane vesicles (Bérczi and Møller, 2000) is in favour of such an hypothesis. Apart from the consideration of the development of a protonmotive force, there is the problem of utilizing that protonmotive force to generate ATP. No ATP synthase has been identified in eukaryotic plasma membranes. Furthermore, the ATPase of plasma membranes is a P-type ATPase, an unlikely candidate for ATP synthesis.
When nitrite accumulates, it can also be used by cNR as a substrate to produce NO. The Km of cNR for nitrite is about 100 µM, which is higher than that of Ni-NOR, and the reaction is competitively inhibited by nitrate (Ki, 50 µM) (Rockel et al., 2002). There are controversial statements about the cNR reaction rate with nitrite to produce NO. The rate in legume nodules may even exceed the rate of nitrate reduction to (Dean and Harper, 1988). Others show that the rate of NO formation is only 1–2% of the maximal cNR reaction in leaves (Yamasaki et al., 2001; Rockel et al., 2002; Sakihama et al., 2002). When Ser521 is replaced by Asp, cNR is not phosphorylated and is permanently active. The rates of NO emission from tobacco plants expressing this mutated protein are ten times higher (in darkness and under hypoxia) than those in control plants (Lea et al., 2004) suggesting that the reaction of the native protein is tightly regulated.
NO is an important metabolite, acting as a signal molecule in most biological systems. In particular, it is produced in animals (Bredt and Snyder, 1994) and plants (Dordas et al., 2003b, 2004) during hypoxic stress. NO was shown to be formed in fairly large quantities in both alfalfa root cultures and maize cell cultures under hypoxic stress. In Chlamydomonas reinhardtii cells, the addition of nitrite causes the formation of NO with an NR reaction rate of 60 nmol mg–1 Chl min–1 (Sakihama et al., 2002).
There are other possible, but less significant, sources of nitrate/nitrite reduction and NO formation during hypoxia. In acidic and reducing environments, NO can be formed by non-enzymatic reduction of nitrite to nitrous acid. The latter reacts with ascorbate producing dehydroascorbate and NO (Weitzberg and Lundberg, 1998). It was shown that, in aleurone layers, conditions are favourable for the non-enzymatic formation of NO (Bethke et al., 2004). Xanthine oxidoreductase located in peroxisomes (Corpas et al., 2001) is also an enzyme producing NO (Harrison, 2002). The NO-generating activity of xanthine oxidoreductase with nitrite is increased at low oxygen tensions (Millar et al., 1997; Godber et al., 2000). It is likely that this enzyme produces superoxide at high oxygen concentrations, and NO at low oxygen concentrations.
In animals and micro-organisms, the main source of NO is the enzyme NO synthase (Bredt and Snyder, 1994). The formation of NO during hypoxia via the NO synthase reaction is unlikely, since this enzyme consumes O2. Evidence of the presence of NO synthase in plants has been controversial until recently (Butt et al., 2003), when it was shown that a modified version of the P-protein of the glycine decarboxylase complex exhibits this activity (Chandok et al., 2003). The activity, however, is low and may be present only in tissues such as green leaves where glycine decarboxylase is abundant. Its role, therefore, probably involves signalling during pathogen attack. Another NO synthase involved in hormonal signalling has been identified, with no sequence similarity to any mammalian isoform (Guo et al., 2003).
The rate of NO formation is reported to be in the range of 10–50 nmol g–1 FW h–1 under hypoxic conditions and 0.2–0.5 nmol g–1 FW h–1 under dark, aerobic conditions (Rockel et al., 2002; Dordas et al., 2003b). Ni-NOR rates in roots are above 0.5 (or even 5–10) µmol g–1 FW h–1 (estimated from Stöhr et al., 2001), well above the range necessary to explain the observed NO rates of formation. The ability of plant cells to scavenge NO can be as high as 4–5 µmol NO g–1 FW min–1 measured in root extracts at 1 µM NO concentration (Igamberdiev et al., 2004b), suggesting that measurements of NO formation in situ are likely to be underestimated. It also suggests that there is an effective system to break down NO in plant tissue, which has been proposed to involve haemoglobin (Dordas et al., 2003a). It is also possible that, under anaerobiosis, the mitochondrial cytochrome c oxidase can use NO as an electron acceptor. It has been suggested that the enzyme evolved from the anaerobic NO reductase family (De Vries and Schröder, 2002) and in higher organisms, including mammals, it can exhibit NO oxidase and peroxynitrite reductase activities (Borutaite and Brown, 1996; Pearce et al., 2002). The importance of these activities in plant systems for maintaining anaerobic ATP synthesis has not been investigated. In anoxia-tolerant Echinochloa, there are indications of the possibility of nitrate reduction by mitochondria, similar to that in bacteria using cytochrome d that transfers electrons to nitrate (Fox et al., 1994). A corresponding absorbance band for such a cytochrome was identified, but these investigations have not been continued.
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Role of haemoglobin |
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TopAbstractIntroductionNitrate uptake and anaerobiosisRegulation of nitrate reductases...Role of haemoglobinThe Hb/NO cycle and...ConclusionReferences |
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The expression of a haemoglobin gene accompanying hypoxia was first demonstrated in barley (Taylor et al., 1994
), in conjunction with the initial work on the existence of this type of haemoglobin in monocots. The gene is induced within 2 h under hypoxic conditions and disappears as rapidly after reinstatement of normal atmospheric conditions. The properties of the Hb protein (Duff et al., 1997) indicated that it probably did not function as a carrier, store, or sensor of O2 (Hill, 1998). While the main physiological function of the gene probably relates to conditions of oxygen starvation, Hb gene induction is more directly related to cell ATP status than to oxygen levels (Nie and Hill, 1997). Expression of the gene during hypoxia probably affects cell survival, as overexpression of Hb in hypoxic maize cell cultures resulted in the maintenance of cell energy status (Sowa et al., 1998). Barley class 1 haemoglobin is a homodimer with a monomeric molecular weight of 18 kDa (Duff et al., 1997). Its affinity for O2 is two orders of magnitude higher (2–3 nM) than that of cytochrome c oxidase (140 nM), evidence that it remains oxygenated at extremely low O2 tensions. Other class 1 haemoglobins possess similar properties (Arredondo-Peter et al., 1997; Hargrove et al., 1997; Kundu et al., 2003a). The unique features of class 1 haemoglobins result from the hexaco-ordination of the haem moiety compared with the pentaco-ordination which exists in haemoglobins such as erythrocyte, muscle haemoglobins, and leghaemoglobins. Hexaco-ordinate haemoglobins are capable of reversible intramolecular co-ordination of the ligand binding site by way of an amino acid side chain from within the haem pocket (Trent et al., 2001).
In addition to maintenance of cell energy status during hypoxia, class 1 haemoglobin expression leads to lower NAD(P)H/NAD(P)) ratios within the hypoxic cell (Igamberdiev et al., 2004b). An involvement of haemoglobin in the oxidation of NAD(P)H is very plausible. There are flavohaemoglobins that catalyse the oxygenation of NO, such as in E. coli (Poole and Hughes, 2000) or yeast (Zhu and Riggs, 1992), with the flavin domain providing the catalytic site for oxidation of reduced flavin and, indirectly, reduced pyridine nucleotide. These proteins, with pentaco-ordinated haem, have a relatively high oxygen affinity, compared with plant class 1 haemoglobins, and form nitrate only under aerobic conditions. There is no evidence of a flavin-binding domain in plant haemoglobins. Any analogous system in plants would, therefore, require haemoglobin to react in concert with another protein, either individually or as part of a dioxygenase complex. Oxyhaemoglobin would donate negatively charged dioxygen to NO, forming nitrate and methaemoglobin, a known reaction of oxyhaemoglobin (Di Iorio, 1981). The reduction of methaemoglobin to haemoglobin can occur in a number of ways. A methaemoglobin reductase has been demonstrated in nodules of leguminous plants (Topunov et al., 1980). A number of diaphorase-type enzymes, such as cytochrome b5 reductase of the endoplasmic reticulum (Hagler et al., 1979) or dihydrolipoamide dehydrogenase (Moran et al., 2002; Igamberdiev et al., 2004a), have methaemoglobin reductase activity. Another possibility is the presence of this reaction in the haemoglobin molecule itself.
No example has yet been observed of an organism that has genes or expresses a flavohaemoglobin with hexaco-ordinate haem properties (Kundu et al., 2003b). Cyanobacteria, Chlamydomonas, all plants, and most animal species contain hexaco-ordinate haemoglobins and no flavohaemoglobins. This has led to the suggestion that hexaco-ordinate haemoglobins in these species may serve a similar role as that of flavohaemoglobins in bacteria and yeast (Kundu et al., 2003b). Participation of a truncated Hb which is hexaco-ordinated at alkaline pH is likely in NO scavenging in Chlamydomonas (Couture et al., 1999).
In micro-organisms, NO is scavenged by NO dioxygenase (NOD), which is a flavohaemoglobin possessing NAD(P)H-dependent enzymatic activity (Gardner et al., 1998, 2000). The NOD reaction is described by the equation
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), however, the identity of a particular haemoglobin and flavin remains undetermined.
Anoxic maize cells overexpressing class 1 haemoglobin have lower ADH activity compared with the wild type and lines down-regulating haemoglobin (Sowa et al., 1998). Higher haemoglobin levels would result in a greater turnover of NO in the Hb/NO cycle, which oxidizes NADH (Fig. 1), replacing to some extent the requirement for ADH activity. Lower NADH/NAD and NADPH/NADP ratios in plants overexpressing haemoglobin have been observed (Igamberdiev et al., 2004b). These ratios are not affected significantly by hypoxia in Hb-overexpressing lines, while in the lines down-regulating Hb the ratios increase drastically under low oxygen tensions. It is evident that the expression of haemoglobin in hypoxic cells, in addition to maintaining energy status, also assists in maintaining the redox status of the cell. Whether it does so solely through the fermentation pathways and the operation of an Hb/NO cycle or through a membrane-associated electron transport is an unresolved question.
Strong hypoxic induction of the Hb gene, comparable with the induction of alcohol dehydrogenase, occurs in Arabidopsis root cultures in concert with the induction of enzymes of nitrogen metabolism, including nitrate reductases (especially nitrate reductase-2 which may be PM-NR) (Klok et al., 2002). Several protein kinases have a similar profile of induction (Klok et al., 2002) indicating a possible link between decreasing ATP levels and Hb synthesis (Nie and Hill, 1997). Hb induction is also observed in response to nitrate (Nie and Hill, 1997), nitrite, and NO treatment (Ohwaki et al., 2003).
Ascaris hexaco-ordinated haemoglobin reacts with NO to produce nitrate, with the oxidation of NADPH, presumably without participation of any additional protein (Minning et al., 1999). Some NADH-dependent NO-degrading activity has also been demonstrated for bacterial haemoglobins lacking a flavoprotein subunit or corresponding domain (Frey et al., 2002). This activity is increased 4–5-fold by the insertion of the reductase domain. Even leghaemoglobin can be reduced non-enzymatically by NADH, reduced glutathione, or ascorbate, but with a lower rate than with the help of methaemoglobin reductase (Becana and Klucas, 1990). Since the above studies use relatively large concentrations of haemoglobin, from an enzyme standpoint, to demonstrate that the haemoglobin has NO dioxygenase activity, the results must be treated with caution. A small impurity in the haemoglobin preparation that possesses reductase activity could account for all of the observed activity. The authors' own unpublished studies with a mutant barley Hb, in which the only cysteine in the monomer has been modified to a serine (Cys79 to Ser), have demonstrated that the NO-dioxygenase activity associated with the mutant haemoglobin involves the participation of a component that is sensitive to sulphydryl reagents, indicating that Hb alone is incapable of sustaining physiologically significant NAD(P)H-dependent NO-degrading activity.
The expression of a class 1 haemoglobin has a direct effect on the level of NO found under hypoxic conditions. Transformed plant tissues (alfalfa roots, maize cells) overexpressing haemoglobin have approximately half the NO levels of control plants under hypoxic conditions, whereas those underexpressing the protein have about twice the NO levels of control plants (Dordas et al., 2003b, 2004; Igamberdiev et al., 2004b). Cytoplasmic extracts of alfalfa root cultures have NO dioxygenase activity that is dependent on haemoglobin and NAD(P)H, thus supporting the mechanism proposed in Fig. 1. The activity exhibited a broad pH optimum and a strong affinity to NADH and NADPH, with a Km=3 µM for both nucleotides (Igamberdiev et al., 2004b).
The evidence presented here indicates that class 1 haemoglobins modulate NO levels within the plant cell. From the mounting data of the involvement of NO in many signal transduction pathways (Wendehenne et al., 2001; Desikan et al., 2002; Hoeberichts and Woltering, 2003; Neill et al., 2003), it is apparent that the presence of this molecule would severely interfere with signal transduction. This, therefore, provides a reasonable argument as to why this gene is only expressed under specific conditions in which cell energy levels are depressed.
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The Hb/NO cycle and carbon metabolism |
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TopAbstractIntroductionNitrate uptake and anaerobiosisRegulation of nitrate reductases...Role of haemoglobinThe Hb/NO cycle and...ConclusionReferences |
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A major unanswered question is how Hb induction during hypoxia maintains plant energy status. An obvious route is via increased substrate level phosphorylation through increased glycolytic flux (Dordas et al., 2003). Figure 3 summarizes the sequence of events resulting in the induction of Hb during plant adaptation to hypoxia. Oxygen deficiency causes a decrease of mitochondrial respiration, which is partly compensated by increased glycolytic flux. As a result, NADH levels increase and ATP levels decrease. ATP decline causes nitrate reductase activation (Stöhr and Mäck, 2001
) and Hb gene induction (Nie and Hill, 1997). Upon the accumulation of nitrite, NO production increases, and Hb participates in NO oxidation to nitrate. Associated with the oxidation of NADH and the Hb/NO cycle during hypoxia/anoxia is the maintenance of the redox and energy status of the cell. In their study of the products of anaerobic nitrate and ammonium ion metabolism in rice coleoptiles, Fan et al. (1997) found an approximately 55% higher production of glycolytic products in the presence of ammonium ion compared with nitrate under anaerobic conditions. This would favour an argument that the observed effects on NO, ATP, and NADH levels during hypoxia in plants overexpressing haemoglobin are not the result of increases in glycolytic flux. This type of study requires revisiting under conditions where the expression of Hb is controlled to determine what part glycolytic flux plays in the overall process.
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Evidence has been accumulating of a putative nitrate respiratory pathway, capable of generating a proton motive force, in bacteria (Jormakka et al., 2003
). The presence of such a pathway in plants, potentially generating ATP more efficiently, might explain why Fan et al. (1997) observe less carbon flow under nitrate than under ammonium under anoxia. This, however, is remote since the known ATPases present in plasma membrane fractions are not of a type that are capable of utilizing chemiosmotic gradients. There is clearly a need for further study to unravel the mechanism by which haemoglobin overexpression results in enhanced energy status in anoxic plants.
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Conclusion |
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TopAbstractIntroductionNitrate uptake and anaerobiosisRegulation of nitrate reductases...Role of haemoglobinThe Hb/NO cycle and...ConclusionReferences |
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A case has been presented here for the role of nitrate, NO, and haemoglobin in maintaining plant cell viability under anoxic stress. It is proposed that this process is an essential part of the acclimation of plants to hypoxic conditions, disappearing once avoidance mechanisms, such as aerenchyma formation, are in place. Nitrate can be viewed as an intermediate electron acceptor under conditions of oxygen deficiency. NO is formed from nitrite by nitrate reductase. NO is oxygenated by a hypoxically-induced class 1 haemoglobin with an extremely high oxygen avidity. The turnover of this reaction is maintained by a linkage to a methaemoglobin reductase, probably inherent in a separate flavoprotein. This cyclic reaction helps to maintain the redox status of the cell at very low oxygen tensions and may reduce the need for fermentative pathways to generate glycolytic energy (Figs 1, 3).
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Acknowledgements |
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This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGP4689) and Genome Canada. We thank Doug Durnin for help in the preparation of the figures.
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Footnotes |
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Abbreviations: cNR, cytosolic nitrate reductase; Hb, haemoglobin; Ni-NOR, nitrite-NO reductase; NO, nitric oxide; PM-NR, plasma membrane-bound nitrate reductase.
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TopAbstractIntroductionNitrate uptake and anaerobiosisRegulation of nitrate reductases...Role of haemoglobinThe Hb/NO cycle and...ConclusionReferences |
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