Non-symbiotic Hemoglobin Research Articles

Nitric oxide enhances the nitrate stress tolerance of spinach by scavenging ROS and RNS

In this study, the role of nitric oxide (NO) in response to excess nitrate stress in spinach was investigated. The results showed that excess nitrate stress provoked a significant reduction in the seedling growth and an increase in lipid peroxidation and H2O2 content in spinach. The gene and protein expression of nitrate reductase and non-symbiotic hemoglobin were up-regulated, while S-nitrosoglutathione reductase (GSNOR) was down-regulated with the accumulation of NO and total S-nitrosothiols (SNOs) after 160mM nitrate treatment for 24h. Nitrate stress treatment increased the S-nitrosylation level of spinach. Using biotin-switch coupled with LC–MS/MS method, several proteins were identified as targets of S-nitrosylation including ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, phosphoglycerate kinase, 23kDa oxygen-evolving protein (OEC), 16kDa protein of the photosynthetic OEC, and ribulose-1,5-bisphosphate carboxylase oxygenase. We further demonstrated that exogenous NO donor (SNP) increased the superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and GSNOR activities, which would reduce reactive oxygen species (ROS) and reactive nitrogen species (RNS) contents and thereby enhance spinach tolerance to nitrate stress. Suppressing NO accumulation by NR inhibitor, NOS inhibitor, and NO scavenger aggravated the damage to spinach under nitrate stress. These results suggest that NO acts as an essential signal to enhance spinach tolerance to nitrate stress via reducing ROS and RNS toxicity.

Nitric oxide-fixation by non-symbiotic haemoglobin proteins in Arabidopsis thaliana under N-limited conditions.

Nitric oxide (NO) is an important signalling molecule that is involved in many different physiological processes in plants. Here, we report about a NO-fixing mechanism in Arabidopsis, which allows the fixation of atmospheric NO into nitrogen metabolism. We fumigated Arabidopsis plants cultivated in soil or as hydroponic cultures during the whole growing period with up to 3 ppmv of NO gas. Transcriptomic, proteomic and metabolomic analyses were used to identify non-symbiotic haemoglobin proteins as key components of the NO-fixing process. Overexpressing non-symbiotic haemoglobin 1 or 2 genes resulted in fourfold higher nitrate levels in these plants compared with NO-treated wild-type. Correspondingly, rosettes size and weight, vegetative shoot thickness and seed yield were 25, 40, 30, and 50% higher, respectively, than in wild-type plants. Fumigation with 250 ppbv 15 NO confirmed the importance of non-symbiotic haemoglobin 1 and 2 for the NO-fixation pathway, and we calculated a daily uptake for non-symbiotic haemoglobin 2 overexpressing plants of 250 mg N/kg dry weight. This mechanism is probably important under conditions with limited N supply via the soil. Moreover, the plant-based NO uptake lowers the concentration of insanitary atmospheric NOx, and in this context, NO-fixation can be beneficial to air quality.

Regulation of programmed cell death by phytoglobins.

Programmed cell death (PCD) is a fundamental plant process in growth and development and in response to both biotic and abiotic stresses. Nitric oxide (NO) is a central component in determining whether a cell undergoes PCD, either as a direct elicitor of the response or as a factor in signal transduction from various hormones. Both NO and hormones that use NO as a signal transducer are mobile in the plant. Why do one set of cells die while adjacent cells remain alive, if this is the case? There is evidence to suggest that phytoglobins (Pgbs; previously termed non-symbiotic hemoglobins) may act as binary switches to determine plant cellular responses to perturbations. There are anywhere from one to five Pgb genes in plants that are expressed in response to growth and development and to stress. One of their main functions is to scavenge NO. This review will discuss how Pgb modulates cellular responses to auxin, cytokinin, and jasmonic acid during growth and development and in response to stress. The moderation in the production of reactive oxygen species (ROS) by Pgbs and the effects on PCD will also be discussed. An overall mechanism for Pgb involvement will be presented.

Overexpression of spinach non-symbiotic hemoglobin in Arabidopsis resulted in decreased NO content and lowered nitrate and other abiotic stresses tolerance.

A class 1 non-symbiotic hemoglobin family gene, SoHb, was isolated from spinach. qRT-PCR showed that SoHb was induced by excess nitrate, polyethylene glycol, NaCl, H2O2, and salicylic acid. Besides, SoHb was strongly induced by application of nitric oxide (NO) donor, while was suppressed by NO scavenger, nitrate reductase inhibitor, and nitric oxide synthase inhibitor. Overexpression of SoHb in Arabidopsis resulted in decreased NO level and sensitivity to nitrate stress, as shown by reduced root length, fresh weight, the maximum photosystem II quantum ratio of variable to maximum fluorescence (Fv/Fm), and higher malondialdehyde contents. The activities and gene transcription of superoxide dioxidase, and catalase decreased under nitrate stress. Expression levels of RD22, RD29A, DREB2A, and P5CS1 decreased after nitrate treatment in SoHb-overexpressing plants, while increased in the WT plants. Moreover, SoHb-overexpressing plants showed decreased tolerance to NaCl and osmotic stress. In addition, the SoHb-overexpression lines showed earlier flower by regulating the expression of SOC, GI and FLC genes. Our results indicated that the decreasing NO content in Arabidopsis by overexpressing SoHb might be responsible for lowered tolerance to nitrate and other abiotic stresses.

Reactive Nitrogen Species in Mitochondria and Their Implications in Plant Energy Status and Hypoxic Stress Tolerance.

Hypoxic and anoxic conditions result in the energy crisis that leads to cell damage. Since mitochondria are the primary organelles for energy production, the support of these organelles in a functional state is an important task during oxygen deprivation. Plant mitochondria adapted the strategy to survive under hypoxia by keeping electron transport operative even without oxygen via the use of nitrite as a terminal electrons acceptor. The process of nitrite reduction to nitric oxide (NO) in the mitochondrial electron transport chain recycles NADH and leads to a limited rate of ATP production. The produced ATP alongside with the ATP generated by fermentation supports the processes of transcription and translation required for hypoxic survival and recovery of plants. Non-symbiotic hemoglobins (called phytoglobins in plants) scavenge NO and thus contribute to regeneration of NAD+ and nitrate required for the operation of anaerobic energy metabolism. This overall operation represents an important strategy of biochemical adaptation that results in the improvement of energy status and thereby in protection of plants in the conditions of hypoxic stress.

Structural and Functional Significance of the N- and C-Terminal Appendages in Arabidopsis Truncated Hemoglobin.

Plant hemoglobins constitute three distinct groups: symbiotic, nonsymbiotic, and truncated hemoglobins. Structural investigation of symbiotic and nonsymbiotic (class I) hemoglobins revealed the presence of a vertebrate-like 3/3 globin fold in these proteins. In contrast, plant truncated hemoglobins are similar to bacterial truncated hemoglobins with a putative 2/2 α-helical globin fold. While multiple structures have been reported for plant hemoglobins of the first two categories, for plant truncated globins only one structure has been reported of late. Here, we report yet another crystal structure of the truncated hemoglobin from Arabidopsis thaliana (AHb3) with two water molecules in the heme pocket, of which one is distinctly coordinated to the heme iron, unlike the only available crystal structure of AHb3 with a hydroxyl ligand. AHb3 was monomeric in its crystallographic asymmetric unit; however, dimer was evident in the crystallographic symmetry, and the globin indeed existed as a stable dimer in solution. The tertiary structure of the protein exhibited a bacterial-like 2/2 α-helical globin fold with an additional N-terminal α-helical extension and disordered C-termini. To address the role of these extended termini in AHb3, which is yet unknown, N- and C-terminal deletion mutants were created and characterized and molecular dynamics simulations performed. The C-terminal deletion had an insignificant effect on most properties but perturbed the dimeric equilibrium of AHb3 and significantly influenced azide binding kinetics in the ferric state. These results along with the disordered nature of the C-terminus indicated its putative role in intramolecular or intermolecular interactions probably regulating protein-ligand and protein-protein interactions. While the N-terminal deletion did not change the overall globin fold, stability, or ligand binding kinetics, it seemed to have influenced coordination at the heme iron, the hydration status of the active site, and the quaternary structure of AHb3. Evidence indicated that the N-terminus is the predominant factor regulating the quaternary interaction appropriate to physiological requirements, dynamics of the side chains in the heme pocket, and tunnel organization in the protein matrix.

Effect of the synthesis of rice non-symbiotic hemoglobins 1 and 2 in the recombinant Escherichia coli TB1 growth

Non-symbiotic hemoglobins (nsHbs) are widely distributed in land plants, including rice. These proteins are classified into type 1 (nsHbs-1) and type 2. The O 2-affinity of nsHbs-1 is very high mostly because of an extremely low O 2-dissociation rate constant resulting in that nsHbs-1 apparently do not release O 2 after oxygenation. Thus, it is possible that the in vivo function of nsHbs-1 is other than O 2-transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however the in vivo activity of rice nsHbs-1 has been poorly analyzed. An in vivo analysis for rice nsHbs-1 is essential to elucidate the biological function(s) of these proteins. Rice Hb1 and Hb2 are nsHbs-1 that have been generated in recombinant Es cherichia coli TB1. The rice Hb1 and Hb2 amino acid sequence, tertiary structure and rate and equilibrium constants for the reaction of O 2 are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarly in vivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinant E. coli TB1 growth. Effect of the synthesis of the O 2-carrying soybean leghemoglobin a, cowpea leghemoglobin II and Vitreoscilla Hb in the recombinant E. coli TB1 growth was also analyzed as an O 2-carrier control. Our results showed that synthesis of rice Hb1, rice Hb2, soybean Lb a, cowpea LbII and Vitreoscilla Hb inhibits the recombinant E. coli TB1 growth and that growth inhibition was stronger when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1. These results suggested that rice Hb1 and Hb2 could function differently in vivo.

Effect of the synthesis of rice non-symbiotic hemoglobins 1 and 2 in the recombinant Escherichia coli TB1 growth

Non-symbiotic hemoglobins (nsHbs) are widely distributed in land plants, including rice. These proteins are classified into type 1 (nsHbs-1) and type 2. The O 2-affinity of nsHbs-1 is very high mostly because of an extremely low O 2-dissociation rate constant resulting in that nsHbs-1 apparently do not release O 2 after oxygenation. Thus, it is possible that the in vivo function of nsHbs-1 is other than O 2-transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however the in vivo activity of rice nsHbs-1 has been poorly analyzed. An in vivo analysis for rice nsHbs-1 is essential to elucidate the biological function(s) of these proteins. Rice Hb1 and Hb2 are nsHbs-1 that have been generated in recombinant Es cherichia coli TB1. The rice Hb1 and Hb2 amino acid sequence, tertiary structure and rate and equilibrium constants for the reaction of O 2 are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarly in vivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinant E. coli TB1 growth. Effect of the synthesis of the O 2-carrying soybean leghemoglobin a, cowpea leghemoglobin II and Vitreoscilla Hb in the recombinant E. coli TB1 growth was also analyzed as an O 2-carrier control. Our results showed that synthesis of rice Hb1, rice Hb2, soybean Lb a, cowpea LbII and Vitreoscilla Hb inhibits the recombinant E. coli TB1 growth and that growth inhibition was stronger when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1. These results suggested that rice Hb1 and Hb2 could function differently in vivo.

Effect of the synthesis of rice non-symbiotic hemoglobins 1 and 2 in the recombinant Escherichia coli TB1 growth

Non-symbiotic hemoglobins (nsHbs) are widely distributed in land plants, including rice. These proteins are classified into type 1 (nsHbs-1) and type 2. The O2-affinity of nsHbs-1 is very high mostly because of an extremely low O2-dissociation rate constant resulting in that nsHbs-1 apparently do not release O2after oxygenation. Thus, it is possible that theinvivofunction of nsHbs-1 is other than O2-transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however theinvivoactivity of rice nsHbs-1 has been poorly analyzed. Aninvivoanalysis for rice nsHbs-1 is essential to elucidate the biological function(s) of these proteins. Rice Hb1 and Hb2 are nsHbs-1 that have been generated in recombinantEscherichiacoliTB1. The rice Hb1 and Hb2 amino acid sequence, tertiary structure and rate and equilibrium constants for the reaction of O2are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarlyinvivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinantE.coliTB1 growth. Effect of the synthesis of the O2-carrying soybean leghemoglobina, cowpea leghemoglobin II andVitreoscillaHb in the recombinantE.coliTB1 growth was also analyzed as an O2-carrier control. Our results showed that synthesis of rice Hb1, rice Hb2, soybean Lba, cowpea LbII andVitreoscillaHb inhibits the recombinantE.coliTB1 growth and that growth inhibition was stronger when recombinantE.coliTB1 synthesized rice Hb2 than when synthesized rice Hb1. These results suggested that rice Hb1 and Hb2 could function differentlyin vivo.

Non-symbiotic hemoglobin and its relation with hypoxic stress

Today we know that several types of hemoglobins exist in plants. The symbiotic hemoglobins were discovered in 1939 and are only found in nodules of plants capable of symbiotically fixing atmospheric N. Another class, called non-symbiotic hemoglobin, was discovered 32 yr ago and is now thought to exist throughout the plant kingdom, being expressed in different organs and tissues. Recently the existence of another type of hemoglobin, called truncated hemoglobin, was demonstrated in plants. Although the presence of hemoglobins is widespread in the plant kingdom, their role has not yet been fully elucidated. This review discusses recent findings regarding the role of plant hemoglobins, with special emphasis on their relationship to plants adaptation to hypoxia. It also discusses the role of nitric oxide in plant cells under hypoxic conditions, since one of the functions of hemoglobin appears to be modulating nitric oxide levels in the cells.

Characterization of unusual truncated hemoglobins of Chlamydomonas reinhardtii suggests specialized functions.

Annotated hemoglobin genes in Chlamydomonas reinhardtii form functional globins, despite unusual architectures. Spectral characteristics show subtle biochemical differences. Multiple globins might help the alga to cope with its versatile environment. The unicellular green alga C. reinhardtii is a photosynthetic, often soil-dwelling organism, subjected to a changeable environment in nature. The alga contains 12 genes encoding so-called truncated hemoglobins that feature a two-on-two helical fold instead of the three-on-three helix arrangement of the long-studied vertebrate globins or plant symbiotic and non-symbiotic hemoglobins. In plants, non-symbiotic hemoglobins often play a role in acclimation to stress, and we could show recently that one of the C. reinhardtii globin genes is vital for anoxic growth. Here, three further globin encoding transcripts (Cre16.g661000.t1.1, Cre16.g661300.t2.1 and Cre16.g662750.t1.2) were heterologously expressed along with the recently studied THB1. UV-Vis and X-ray absorption spectroscopy analyses show that the sequences indeed encode functional hemoglobins, despite their uncommon primary sequences, which include long C-termini without any predictable function, or a split heme-binding domain. The proteins show some variations regarding the coordination of the heme iron or the interaction with diatomic ligands, indicating different functionalities. The respective transcripts are not responsive to the nitrogen source, in contrast to results reported for THB1, but they accumulate in darkness. This work advances experimental data on the very large globin family in general, and, more specifically, on hemoglobins in photosynthetic organisms.

Role of Ionic Strength and the Bohr Effect in Modulating Thermodynamic Profiles Associated with Co Escape in Rice Non-Symbiotic Hemoglobin 1

Rice hemoglobin (rHb1) is a type 1 non-symbiotic hemoglobin (nsHb1), which is found in plants and which is evolutionarily related to leghemoglobins and truncated hemoglobins. It is structurally similar to vertebrate heme proteins, containing Fe-protoporphyrin IX and the classic 3-over-3 globin fold. Unlike other hexacoordinate heme proteins found in plants, the ligand binding properties of nsHb1 proteins make it unlikely to be involved in nitrogen fixation or oxygen transport. We have characterized ligand migration upon CO photorelease from rHb1 under various conditions in order to elucidate functional role(s) of rHb1 in vivo. Using photoacoustic calorimetry, we have determined thermodynamic parameters (ΔH and ΔV) for CO photorelease from wild type rHb1 and a distal histidine to leucine (H74L) mutant taking place within ∼ 5 µs after ligand photo-dissociation. Under stripped conditions, we observe a strong temperature dependence of the observed parameters. Photorelease of CO from the wild type protein below 16°C is associated with ΔH = 28.7 ± 2.2 kcal mol−1 and ΔV = 4.3 ± 0.4 mL mol−1, while above 16°C we observe ΔH = 9.7 ± 3.4 kcal mol−1 and ΔV = 0.4 ± 0.7 mL mol−1. This temperature dependence is not observed in the H74L mutant, in the presence of 500 mM NaCl, or at pH 6. We have also determined rate constants and quantum yields for bimolecular rebinding of CO using transient absorption spectroscopy, both of which increase with temperature. The contributions of electrostriction, proton uptake by Bohr groups, oligomerization state, and the distal residue of rHb1 to the observed kinetic and thermodynamic parameters will be discussed. We will also discuss ligand migration pathways and protein dynamics upon CO photorelease in rHb1.

De novo sequencing, assembly, and analysis of the Taxodium 'Zhongshansa' roots and shoots transcriptome in response to short-term waterlogging.

BackgroundTaxodium is renowned for its strong tolerance to waterlogging stress, thus it has great ecological and economic potential. However, the scant genomic resources in genus Taxodium have greatly hindered further exploration of its underlying flood-tolerance mechanism. Taxodium ‘Zhongshansa’ is an interspecies hybrid of T. distichum and T. mucronatum, and has been widely planted in southeastern China. To understand the genetic basis of its flood tolerance, we analyzed the transcriptomes of Taxodium ‘Zhongshansa’ roots and shoots in response to short-term waterlogging.ResultsRNA-seq was used to analyze genome-wide transcriptome changes of Taxodium ‘Zhongshansa 406’ clone root and shoot treated with 1 h of soil-waterlogging stress. After de novo assembly, 108,692 unigenes were achieved, and 70,260 (64.64%) of them were annotated. There were 2090 differentially expressed genes (DEGs) found in roots and 394 in shoots, with 174 shared by both of them, indicating that the aerial parts were also affected. Under waterlogging stress, the primary reaction of hypoxic-treated root was to activate the antioxidative defense system to prevent cells experiencing reactive oxygen species (ROS) poisoning. As respiration was inhibited and ATP decreased, another quick coping mechanism was repressing the energy-consuming biosynthetic processes through the whole plant. The glycolysis and fermentation pathway was activated to maintain ATP production in the hypoxic root. Constantly, the demand for carbohydrates increased, and carbohydrate metabolism were accumulated in the root as well as the shoot, possibly indicating that systemic communications between waterlogged and non-waterlogged tissues facilated survival. Amino acid metabolism was also greatly influenced, with down-regulation of genes involvedin serine degradation and up-regulation of aspartic acid degradation. Additionally, a non-symbiotic hemoglobin class 1 gene was up-regulated, which may also help the ATP production. Moreover, the gene expression pattern of 5 unigenes involving in the glycolysis pathway revealed by qRT-PCR confirmed the RNA-Seq data.ConclusionsWe conclude that ROS detoxification and energy maintenance were the primary coping mechanisms of ‘Zhongshansa’ in surviving oxygen deficiency, which may be responsible for its remarkable waterlogging tolerance. Our study not only provided the first large-scale assessment of genomic resources of Taxodium but also guidelines for probing the molecular mechanism underlying ‘Zhongshansa’ waterlogging tolerance.

Nitric oxide production and scavenging in waterlogged roots of rape seedlings

When plant roots are waterlogged, plants can experience hypoxic stress. Large quantities of nitric oxide (NO) can be generated under hypoxic conditions as a result of nitrate reduction. Our objective was to investigate the molecular mechanisms behind NO production and scavenging (turnover) in waterlogged roots of rape (‘Tammi’ variety) seedlings by surveying waterlogging-responsive genes. Waterlogging for up to 72 h enhanced NO production rapidly in the roots. Of 53,107 genes assayed, 9,692 showed a twofold change in expression within 36 h of waterlogging. Two nitrate reductase (NaR) genes (TC201891, TC161540) and four nitrite reductase (NiR) genes (TC168889, TC164215, TC163914, TC185634) were potentially involved in NO production in response to waterlogging stress. Strong hypoxic induction of non-symbiotic hemoglobin (Hb) gene (TC165566), which increased 656- and 645-fold at 36 and 72 h of waterlogging, respectively, could oxidize the NO overproduced in the roots. Our results suggested that reduction of nitrate to NO by NaR and NiR and subsequent NO turnover by Hb provide a mechanism for maintaining bioenergetics in waterlogged roots. The up-regulation of many additional waterlogging-responsive genes with potential roles in the anaerobic respiration, sucrose and starch degradation, glycolysis, and pyruvate metabolism, may acclimate the plant to waterlogging-induced hypoxic condition.

The structure of a class 3 nonsymbiotic plant haemoglobin from Arabidopsis thaliana reveals a novel N-terminal helical extension.

Plant nonsymbiotic haemoglobins fall into three classes, each with distinct properties but all with largely unresolved physiological functions. Here, the first crystal structure of a class 3 nonsymbiotic plant haemoglobin, that from Arabidopsis thaliana, is reported to 1.77 Å resolution. The protein forms a homodimer, with each monomer containing a two-over-two α-helical domain similar to that observed in bacterial truncated haemoglobins. A novel N-terminal extension comprising two α-helices plays a major role in the dimer interface, which occupies the periphery of the dimer-dimer face, surrounding an open central cavity. The haem pocket contains a proximal histidine ligand and an open sixth iron-coordination site with potential for a ligand, in this structure hydroxide, to form hydrogen bonds to a tyrosine or a tryptophan residue. The haem pocket appears to be unusually open to the external environment, with another cavity spanning the entrance of the two haem pockets. The final 23 residues of the C-terminal domain are disordered in the structure; however, these domains in the functional dimer are adjacent and include the only two cysteine residues in the protein sequence. It is likely that these residues form disulfide bonds in vitro and it is conceivable that this C-terminal region may act in a putative complex with a partner molecule in vivo.

Differential Expression Patterns of Non-Symbiotic Hemoglobins in Sugar Beet (Beta vulgaris ssp. vulgaris)

Biennial sugar beet (Beta vulgaris spp. vulgaris) is a Caryophyllidae that has adapted its growth cycle to the seasonal temperature and daylength variation of temperate regions. This is the first time a holistic study of the expression pattern of non-symbiotic hemoglobins (nsHbs) is being carried out in a member of this group and under two essential environmental conditions for flowering, namely vernalization and length of photoperiod. BvHb genes were identified by sequence homology searches against the latest draft of the sugar beet genome. Three nsHb genes (BvHb1.1, BvHb1.2 and BvHb2) and one truncated Hb gene (BvHb3) were found in the genome of sugar beet. Gene expression profiling of the nsHb genes was carried out by quantitative PCR in different organs and developmental stages, as well as during vernalization and under different photoperiods. BvHb1.1 and BvHb2 showed differential expression during vernalization as well as during long and short days. The high expression of BvHb2 indicates that it has an active role in the cell, maybe even taking over some BvHb1.2 functions, except during germination where BvHb1.2 together with BvHb1.1-both Class 1 nsHbs-are highly expressed. The unprecedented finding of a leader peptide at the N-terminus of BvHb1.1, for the first time in an nsHb from higher plants, together with its observed expression indicate that it may have a very specific role due to its suggested location in chloroplasts. Our findings open up new possibilities for research, breeding and engineering since Hbs could be more involved in plant development than previously was anticipated.

Molecular cloning and protein folding characterization of a predicted heme-binding globin in a sugar cane EST database.

A very large and representative sugar cane expression sequence tag (EST) library (SUCEST) was sequenced by a Brazilian consortium, opening the possibility to study important proteins, such as hemoglobins, which are largely present across the plant kingdom. The widespread presence and long evolutionary history of plant hemoglobins suggest a major role for this protein family in plants; however, little is known about their functional roles. In this study, we report the identification and characterization of a putative non-symbiotic hemoglobin cDNA clone that was identified in SUCEST. The cDNA was cloned, and the recombinant protein was purified and folded, as shown by its circular dichroism and emission fluorescence spectra. The expressed globin protein was able to bind hemin, as a characteristic Soret band was observed in the absorbance spectrum and increases were seen in the amount of secondary structure and in the stability of the protein. A model for the structure of the sugarcane hemoglobin was created using the crystal structure of a rice hemoglobin, and this model showed a conserved globular conformation.

Nitrogen metabolism in plants under low oxygen stress

More frequent flooding and waterlogging events due to more heavy precipitation are expected worldwide in the context of climate change. Accordingly, adaptation of plants to oxygen limitation at both cellular and whole plant levels should be investigated thoroughly, that derived knowledge could be taken into account in breeding programs and agronomical practices for saving plant fitness, growth and development even when oxygen availability is low. In the present review, we highlight current knowledge on essential aspects of low oxygen stress-induced changes in nitrogen metabolism. The involvement of two possible pathways for NO production either via the reaction catalyzed by nitrate reductase or at Complex III or IV of the mitochondrial electron transport chain, thus contributing to ATP synthesis via the so-called nitrite-NO respiration, is discussed. NO is proposed to be scavenged by non-symbiotic hemoglobin (Hb) in a Hb/NO cycle, in which NAD(P)H is oxidized for the conversion of NO into NO3(-). The investigation of an additional adaptation to the decrease in oxygen availability via transcriptional and posttranslational regulation of amino acid synthesis pathways, using publicly available transcriptome and translatome data for Arabidopsis thaliana and rice is also discussed.

Nitric Oxide in Plants: The Roles of Ascorbate and Hemoglobin

Ascorbic acid and hemoglobins have been linked to nitric oxide metabolism in plants. It has been hypothesized that ascorbic acid directly reduces plant hemoglobin in support of NO scavenging, producing nitrate and monodehydroascorbate. In this scenario, monodehydroascorbate reductase uses NADH to reduce monodehydroascorbate back to ascorbate to sustain the cycle. To test this hypothesis, rates of rice nonsymbiotic hemoglobin reduction by ascorbate were measured directly, in the presence and absence of purified rice monodehydroascorbate reductase and NADH. Solution NO scavenging was also measured methodically in the presence and absence of rice nonsymbiotic hemoglobin and monodehydroascorbate reductase, under hypoxic and normoxic conditions, in an effort to gauge the likelihood of these proteins affecting NO metabolism in plant tissues. Our results indicate that ascorbic acid slowly reduces rice nonsymbiotic hemoglobin at a rate identical to myoglobin reduction. The product of the reaction is monodehydroascorbate, which can be efficiently reduced back to ascorbate in the presence of monodehydroascorbate reductase and NADH. However, our NO scavenging results suggest that the direct reduction of plant hemoglobin by ascorbic acid is unlikely to serve as a significant factor in NO metabolism, even in the presence of monodehydroascorbate reductase. Finally, the possibility that the direct reaction of nitrite/nitrous acid and ascorbic acid produces NO was measured at various pH values mimicking hypoxic plant cells. Our results suggest that this reaction is a likely source of NO as the plant cell pH drops below 7, and as nitrite concentrations rise to mM levels during hypoxia.

MiR172 Regulates Soybean Nodulation

Micro-RNAs (miRNAs) play a pivotal role in the control of gene expression and regulate plant developmental processes. miRNA 172 (miR172) is a conserved miRNA in plants reported to control the expression of genes involved in developmental phase transition, floral organ identity, and flowering time. However, the specific role of miR172 in legume nodulation is undefined. Ectopic expression of soybean miR172 resulted in an increase in nodule numbers in transgenic roots and an increase in the expression of both symbiotic leghemoglobin and nonsymbiotic hemoglobin. These nodules showed higher levels of nitrogenase activity. Further analysis revealed a complex regulatory circuit in which miR156 regulates miR172 expression and controls the level of an AP2 transcription factor. The latter, either directly or indirectly, controls the expression of nonsymbiotic hemoglobin, which is essential for regulating the levels of nodulation.