Non-symbiotic Hemoglobin Research Articles

Bicarbonate-Dependent Detoxification by Mitigating Ammonium-Induced Hypoxic Stress in Triticum aestivum Root.

Ammonium (NH4+) toxicity is ubiquitous in plants. To investigate the underlying mechanisms of this toxicity and bicarbonate (HCO3-)-dependent alleviation, wheat plants were hydroponically cultivated in half-strength Hoagland nutrient solution containing 7.5 mM NO3- (CK), 7.5 mM NH4+ (SA), or 7.5 mM NH4+ + 3 mM HCO3- (AC). Transcriptomic analysis revealed that compared to CK, SA treatment at 48 h significantly upregulated the expression of genes encoding fermentation enzymes (pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH)) and oxygen consumption enzymes (respiratory burst oxidase homologs, dioxygenases, and alternative oxidases), downregulated the expression of genes encoding oxygen transporters (PIP-type aquaporins, non-symbiotic hemoglobins), and those involved in energy metabolism, including tricarboxylic acid (TCA) cycle enzymes and ATP synthases, but upregulated the glycolytic enzymes in the roots and downregulated the expression of genes involved in the cell cycle and elongation. The physiological assay showed that SA treatment significantly increased PDC, ADH, and LDH activity by 36.69%, 43.66%, and 61.60%, respectively; root ethanol concentration by 62.95%; and lactate efflux by 23.20%, and significantly decreased the concentrations of pyruvate and most TCA cycle intermediates, the complex V activity, ATP content, and ATP/ADP ratio. As a consequence, SA significantly inhibited root growth. AC treatment reversed the changes caused by SA and alleviated the inhibition of root growth. In conclusion, NH4+ treatment alone may cause hypoxic stress in the roots, inhibit energy generation, suppress cell division and elongation, and ultimately inhibit root growth, and adding HCO3- remarkably alleviates the NH4+-induced inhibitory effects on root growth largely by attenuating the hypoxic stress.

Dynamics of hemoglobins during nodule development, nitrate response, and dark stress in Lotus japonicus.

Legume nodules express multiple leghemoglobins (Lbs) and non-symbiotic hemoglobins (Glbs), but how they are regulated is unclear. Here, we study the regulation of all Lbs and Glbs of Lotus japonicus in different physiologically relevant conditions and mutant backgrounds. We quantified hemoglobin expression, localized reactive oxygen species (ROS) and nitric oxide (NO) in nodules, and deployed mutants deficient in Lbs and in the transcription factors NLP4 (associated with nitrate sensitivity) and NAC094 (associated with senescence). Expression of Lbs and class 2 Glbs was suppressed by nitrate, whereas expression of class 1 and 3 Glbs was positively correlated with external nitrate concentrations. Nitrate-responsive elements were found in the promoters of several hemoglobin genes. Mutant nodules without Lbs showed accumulation of ROS and NO and alterations of antioxidants and senescence markers. NO accumulation occurred by a nitrate-independent pathway, probably due to the virtual disappearance of Glb1-1 and the deficiency of Lbs. We conclude that hemoglobins are regulated in a gene-specific manner during nodule development and in response to nitrate and dark stress. Mutant analyses reveal that nodules lacking Lbs experience nitro-oxidative stress and that there is compensation of expression between Lb1 and Lb2. They also show modulation of hemoglobin expression by NLP4 and NAC094.

Nitrate-induced AHb1 expression aggravates Cd toxicity in plants

Cadmium (Cd) causes severe toxicity in plants. However, the molecular mechanisms underlying plant resistance to Cd in relation to nitrogen (N) supply remain unclear. The non-symbiotic hemoglobin gene Hb1 plays an important role in scavenging nitric oxide (NO) in plants. In this study, there was no differential effect of Cd on the biomass of wild-type (WT) and AHb1-overexpressing (H7) plants when NH4+-N was used as a nitrogen source. However, under NO3−-N conditions, Cd exerted less biomass stress on AHb1-silenced (L3) plants and more stress on H7 plants than on WT plants. The Cd tolerance index followed the order: L3 > WT > H7. However, there was no difference in Cd concentrations in the roots or shoots of the WT, L3, and H7 plants, indicating that differences in AHb1 expression were unrelated to Cd uptake. Further investigation showed that Cd exposure enhanced H2O2 accumulation and aggravated oxidative damage in H7 plants. The application of an NO donor effectively reversed growth inhibition, H2O2 burst, and oxidative stress induced by Cd in H7 plants. Thus, we suggest that NO3−-induced AHb1 expression suppresses Cd-induced NO production in plants, increasing the ROS burst and exacerbating Cd toxicity.

Peroxidase activity of rice (Oryza sativa) hemoglobin: distinct role of tyrosines 112 and 151.

Five non-symbiotic hemoglobins (nsHb) have been identified in rice (Oryza sativa). Previous studies have shown that stress conditions can induce their overexpression, but the role of those globins is still unclear. To better understand the functions of nsHb, the reactivity of rice Hb1 toward hydrogen peroxide (H2O2) has been studied in vitro. Our results show that recombinant rice Hb1 dimerizes through dityrosine cross-links in the presence of H2O2. By site-directed mutagenesis, we suggest that tyrosine 112 located in the FG loop is involved in this dimerization. Interestingly, this residue is not conserved in the sequence of the five rice non-symbiotic hemoglobins. Stopped-flow spectrophotometric experiments have been performed to measure the catalytic constants of rice Hb and its variants using the oxidation of guaiacol. We have shown that Tyrosine112 is a residue that enhances the peroxidase activity of rice Hb1, since its replacement by an alananine leads to a decrease of guaiacol oxidation. In contrast, tyrosine 151, a conserved residue which is buried inside the heme pocket, reduces the protein reactivity. Indeed, the variant Tyr151Ala exhibits a higher peroxidase activity than the wild type. Interestingly, this residue affects the heme coordination and the replacement of the tyrosine by an alanine leads to the loss of the distal ligand. Therefore, even if the amino acid at position 151 does not participate to the formation of the dimer, this residue modulates the peroxidase activity and plays a role in the hexacoordinated state of the heme.

Possible mechanism of contribution of a secreted aspartic proteinase FpOPSB to the virulence of Fusarium proliferatum causing banana crown rot

AbstractFusarium proliferatum is one of the pathogenic fungi causing crown rot that results in quality deterioration and commercial value loss of banana fruit during postharvest storage. Secreted proteins from pathogenic fungi can be delivered into hosts to suppress plant immunity and establish infection. We reported here a secreted aspartic proteinase OPSB (FpOPSB) that was active during F. proliferatum infection on banana peel. FpOPSB exhibited protease activity based on in vitro assay. Immunohistochemistry assay along with subcellular localization analysis demonstrated that FpOPSB can be secreted into banana peel and localized in cytoplasm. FpOPSB deletion mutant (Δopsb) showed reduced virulence on banana fruit but no obvious effect on fungal growth compared with wild‐type. Subsequently, scanning electron microscopy results suggested that FpOPSB was important for conidial production and infectious growth as well as spreading of F. proliferatum in banana peel. Heterologous expression of FpOPSB in tobacco significantly enhanced plant susceptibility to F. proliferatum. Further, seven proteins were identified as potential targets of FpOPSB in banana peel using immunoprecipitation–mass spectrometry (MS)/MS, including pathogenesis‐related 10, thioredoxin H1, nonsymbiotic hemoglobin 2, fructokinase‐1, patellin‐1, leucine aminopeptidase 2, and l‐lactate dehydrogenase A. Taken together, our results confirmed that the secreted FpOPSB is a critical virulence factor of F. proliferatum when infecting banana fruit. Additionally, we postulated that FpOPSB might interfere the host immune system or neutralize the host defenses, thereby contributing to the pathogenicity of F. proliferatum. We propose aspartic proteinase as a new potential target for controlling fungal disease of postharvest fruit.

Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin.

The recently identified nonsymbiotic hemoglobin gene MtGlb1-2 of the legume Medicago truncatula possesses unique properties as it generates four alternative splice forms encoding proteins with one or two heme domains. Here we investigate the ligand binding kinetics of MtGlb1-2.1 and MtGlb1-2.4, bearing two hemes and one heme, respectively. Unexpectedly, the overall time-course of ligand rebinding was unusually fast. Thus, we complemented nanosecond laser flash photolysis kinetics with data collected with a hybrid femtosecond–nanosecond pump–probe setup. Most photodissociated ligands are rebound geminately within a few nanoseconds, which leads to rates of the bimolecular rebinding to pentacoordinate species in the 108 M−1s−1 range. Binding of the distal histidine to the heme competes with CO rebinding with extremely high rates (kh ~ 105 s−1). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (KH ~ 1). The rate constants for ligation and deligation of distal histidine to the heme are the highest reported for any plant or vertebrate globin. The combination of microscopic rates results in unusually high overall ligand binding rate constants, a fact that contributes to explaining at the mechanistic level the extremely high reactivity of these proteins toward the physiological ligands oxygen, nitric oxide and nitrite.

Structural Insights into the Heme Pocket and Oligomeric State of Non-Symbiotic Hemoglobins from Arabidopsis thaliana.

Non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana are hexacoordinate heme-proteins that likely have different biological roles, in view of diverse tissue localization, expression pattern, and ligand binding properties. Herein, we expand upon previous biophysical studies on these isoforms, focusing on their oligomeric states and circular dichroism (CD) characteristics. We found that AHb1 exists in solution in a concentration-dependent monomer-dimer equilibrium, while AHb2 is present only as a monomer. The quaternary structure of AHb1 affects its degree of hexacoordination with the formation of the dimer that enhances pentacoordination. Accordingly, the mutant of a conserved residue within the dimeric interface, AHb1-T45A, which is mostly monomeric in solution, has an equilibrium that is shifted toward a hexacoordinate form compared to the wild-type protein. CD studies further support differences in the globin’s structure and heme moiety. The Soret CD spectra for AHb2 are opposite in sense to those for AHb1, reflecting different patterns of heme-protein side chain contacts in the two proteins. Moreover, the smaller contribution of the heme to the near-UV CD in AHb2 compared to AHb1 suggests a weaker heme-protein association in AHb2. Our data corroborate the structural diversity of AHb1 and AHb2 and confirm the leghemoglobin-like structural properties of AHb2.

A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215–351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.

Overexpression of BvHb2, a Class 2 Non-Symbiotic Hemoglobin from Sugar Beet, Confers Drought-Induced Withering Resistance and Alters Iron Content in Tomato

Drought stress is one of the major threats to agriculture and concomitantly to food production. Tomato is one of the most important industrial crops, but its tolerance to water scarcity is very low. Traditional plant breeding has a limited margin to minimize this water requirement. In order to design novel biotechnological approaches to cope with this problem, we have screened a plant cDNA library from the halotolerant crop sugar beet (Beta vulgaris L.) for genes able to confer drought/osmotic stress tolerance to the yeast model system upon overexpression. We have identified the gene that encodes BvHb2, a class 2 non-symbiotic hemoglobin, which is present as a single copy in the sugar beet genome, expressed mainly in leaves and regulated by light and abiotic stress. We have evaluated its biotechnological potential in the model plant Arabidopsis thaliana and found that BvHb2 is able to confer drought and osmotic stress tolerance. We also generated transgenic lines of tomato (Solanum lycopersicum) overexpressing BvHb2 and found that the resulting plants are more resistant to drought-induce withering. In addition, transgenic lines overexpressing BvHb2 exhibit increased levels of iron content in leaves. Here, we show that class 2 non-symbiotic plant hemoglobins are targets to generate novel biotechnological crops tolerant to abiotic stress. The fact that these proteins are conserved in plants opens the possibility for using Non-GMO approaches, such as classical breeding, molecular breeding, or novel breeding techniques to increase drought tolerance using this protein as a target.

The role of nitrite and nitric oxide under low oxygen conditions in plants.

Plant tissues, particularly roots, can be subjected to periods of hypoxia due to environmental circumstances. Plants have developed various adaptations in response to hypoxic stress and these have been described extensively. Less well-appreciated is the body of evidence demonstrating that scavenging of nitric oxide (NO) and the reduction of nitrate/nitrite regulate important mechanisms that contribute to tolerance to hypoxia. Although ethylene controls hyponasty and aerenchyma formation, NO production apparently regulates hypoxic ethylene biosynthesis. In the hypoxic mitochondrion, cytochrome c oxidase, which is a major source of NO, also is inhibited by NO, thereby reducing the respiratory rate and enhancing local oxygen concentrations. Nitrite can maintain ATP generation under hypoxia by coupling its reduction to the translocation of protons from the inner side of mitochondria and generating an electrochemical gradient. This reaction can be further coupled to a reaction whereby nonsymbiotic haemoglobin oxidizes NO to nitrate. In addition to these functions, nitrite has been reported to influence mitochondrial structure and supercomplex formation, as well as playing a role in oxygen sensing via the N-end rule pathway. These studies establish that nitrite and NO perform multiple functions during plant hypoxia and suggest that further research into the underlying mechanisms is warranted.

Phytoglobins in the nuclei, cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid.

Symbiotic hemoglobins provide O2 to N2 -fixing bacteria within legume nodules, but the functions of non-symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C-terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S-nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S-nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA-Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.

Analysis of the regulation networks in grapevine reveals response to waterlogging stress and candidate gene-marker selection for damage severity.

Owing to the climate change impacts, waterlogging is one of the most hazardous abiotic stresses to crops, which also can result in a serious reduction in the quantity and quality of grape berry and wine production during the rainy season. Therefore, the exploration of the response mechanism of grape to waterlogging is necessary, for which the analysis of the transcriptomic regulation networks of grapevine leaves in response to waterlogging stress was carried out. In this study, 12 634 genes were detected in both waterlogging stress and control grapevine plants, out of which 6837 genes were differentially expressed. A comparative analysis revealed that genes functioning in the antioxidant system, glycolysis and fermentation pathway, chlorophyll metabolism, amino acid metabolism and hormones were activated to reduce injury to grapes under the waterlogging stress. Meanwhile, genes encoding class-2 non-symbiotic haemoglobin were determined as important in waterlogging acclimation. Additionally, the expression variations of three marker genes were found to be informative and can be used to predict the viability of the grapevines subjected to waterlogging. This research not only probes the molecular mechanism underlying grapevine waterlogging tolerance but also puts forward an idea about the application of gene expression information to practical management.

A discrete role for alternative oxidase under hypoxia to increase nitric oxide and drive energy production

Alternative oxidase (AOX) is an integral part of the mitochondrial electron transport and can prevent reactive oxygen species (ROS) and nitric oxide (NO) production under non-stressed, normoxic conditions. Here we assessed the roles of AOX by imposing stress under normoxia in comparison to hypoxic conditions using AOX over expressing (AOX OE) and anti-sense (AOX AS) transgenic Arabidopsis seedlings and roots. Under normoxic conditions stress was induced with the defence elicitor flagellin (flg22). AOX OE reduced NO production whilst this was increased in AOX AS. Moreover AOX AS also exhibited an increase in superoxide and therefore peroxynitrite, tyrosine nitration suggesting that scavenging of NO by AOX can prevent toxic peroxynitrite formation under normoxia. In contrast, during hypoxia interestingly we found that AOX is a generator of NO. Thus, the NO produced during hypoxia, was enhanced in AOX OE and suppressed in AOX AS. Additionally, treatment of WT or AOX OE with the AOX inhibitor SHAM inhibited hypoxic NO production. The enhanced levels of NO correlated with expression of non-symbiotic haemoglobin, increased NR activity and ATP production. The ATP generation was suppressed in nia1,2 mutant and non symbiotic haemoglobin antisense line treated with SHAM. Taken together these results suggest that hypoxic NO generation mediated by AOX has a discrete role by feeding into the haemoglobin-NO cycle to drive energy efficiency under conditions of low oxygen tension.

Non-symbiotic hemoglobin conformational space dependence on the heme coordination using nESI-TIMS-TOF MS

In this study, for the first time, the conformational space of the rice non-symbiotic hemoglobin type 1 (rHb1) was studied as a function of the starting solution pH using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and molecular dynamics. Comparison of the charge state distribution, apo to holo form ratio, and the collision cross section (Ω) profiles as a function of the solution pH showed higher stability of the rHb1 wild-type (WT) when compared with the H73L mutant at mildly acidic conditions. Comparison of the Ω profiles of the rHb1 WT and H73L holo and apo form showed that only the initial unfolding pathways involved the heme cavity, with and without a heme loss, followed by unfolding pathways not necessarily involving the environment of the heme prosthetic group. Candidate structures for the nine transitions observed in the Ω profiles were proposed using molecular dynamic simulations based on the Ω profiles, UV absorption spectroscopy and circular dichroism data as way to describe a potential unfolding pathway. The described unfolding pathway suggests that the rHb1 unfolding is driven by initial distancing of the A, B, and H helices, while the heme cavity and heme group remains intact, followed by the distancing of the E, F, and G helices and subsequent loss of the α-helical structure leading to a final random coil conformation.

Pathways of nitric oxide metabolism and operation of phytoglobins in legume nodules: missing links and future directions.

The interaction between legumes and rhizobia leads to the establishment of a beneficial symbiotic relationship. Recent advances in legume - rhizobium symbiosis revealed that various reactive oxygen and nitrogen species including nitric oxide (NO) play important roles during this process. Nodule development occurs with a transition from a normoxic environment during the establishment of symbiosis to a microoxic environment in functional nodules. Such oxygen dynamics are required for activation and repression of various NO production and scavenging pathways. Both the plant and bacterial partners participate in the synthesis and degradation of NO. However, the pathways of NO production and degradation as well as their cross-talk and involvement in the metabolism are still a matter of debate. The plant-originated reductive pathways are known to contribute to the NO production in nodules under hypoxic conditions. Non-symbiotic hemoglobin (phytoglobin) (Pgb) possesses high NO oxygenation capacity, buffers and scavenges NO. Its operation, through a respiratory cycle called Pgb-NO cycle, leads to the maintenance of redox and energy balance in nodules. The role of Pgb/NO cycle under fluctuating NO production from soil needs further investigation for complete understanding of NO regulatory mechanism governing nodule development to attain optimal food security under changing environment.

Non-symbiotic Hemoglobin Conformational Space Dependence on the HEME Coordination using NESI-TIMS-TOF MS

In this study, for the first time, the conformational space of the rice non-symbiotic hemoglobin type 1 (rHb1) was studied as a function of the starting solution pH using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and molecular dynamics. Comparison of the charge state distribution, apo to holo form ratio, and the collision cross section (CCS) profiles as a function of the solution pH showed higher stability of the rHb1 wild-type (WT) when compared with the H73L mutant at mildly acidic conditions. Comparison of the CCS profiles of the rHb1 WT and H73L holo and apo form showed that only the initial unfolding pathways involved the heme cavity, with and without a heme loss, followed by unfolding pathways not necessarily involving the environment of the heme prosthetic group. Representative candidate structures for the nine transitions observed in the CCS profiles were proposed using molecular dynamic simulations. Inspection of the representative structures of the CCS ensemble suggests that the rHb1 unfolding is driven by initial distancing of the A, B, and H helices, while the heme cavity and heme group remains intact, followed by the distancing of the E, F, and G helices and subsequent loss of the α-helical structure leading to a final random coil conformation.

Insights into soybean transcriptome reconfiguration under hypoxic stress: Functional, regulatory, structural, and compositional characterization.

Soybean (Glycine max) is one of the major crops worldwide and flooding stress affects the production and expansion of cultivated areas. Oxygen is essential for mitochondrial aerobic respiration to supply the energy demand of plant cells. Because oxygen diffusion in water is 10,000 times lower than in air, partial (hypoxic) or total (anoxic) oxygen deficiency is important component of flooding. Even when oxygen is externally available, oxygen deficiency frequently occurs in bulky, dense or metabolically active tissues such as phloem, meristems, seeds, and fruits. In this study, we analyzed conserved and divergent root transcriptional responses between flood-tolerant Embrapa 45 and flood-sensitive BR 4 soybean cultivars under hypoxic stress conditions with RNA-seq. To understand how soybean genes evolve and respond to hypoxia, stable and differentially expressed genes were characterized structurally and compositionally comparing its mechanistic relationship. Between cultivars, Embrapa 45 showed less up- and more down-regulated genes, and stronger induction of phosphoglucomutase (Glyma05g34790), unknown protein related to N-terminal protein myristoylation (Glyma06g03430), protein suppressor of phyA-105 (Glyma06g37080), and fibrillin (Glyma10g32620). RNA-seq and qRT-PCR analysis of non-symbiotic hemoglobin (Glyma11g12980) indicated divergence in gene structure between cultivars. Transcriptional changes for genes in amino acids and derivative metabolic process suggest involvement of amino acids metabolism in tRNA modifications, translation accuracy/efficiency, and endoplasmic reticulum stress in both cultivars under hypoxia. Gene groups differed in promoter TATA box, ABREs (ABA-responsive elements), and CRT/DREs (C-repeat/dehydration-responsive elements) frequency. Gene groups also differed in structure, composition, and codon usage, indicating biological significances. Additional data suggests that cis-acting ABRE elements can mediate gene expression independent of ABA in soybean roots under hypoxia.

Reduced nitric oxide levels during drought stress promote drought tolerance in barley and is associated with elevated polyamine biosynthesis

Nitric oxide (NO) is a key messenger in plant stress responses but its exact role in drought response remains unclear. To investigate the role of NO in drought response we employed transgenic barley plants (UHb) overexpressing the barley non-symbiotic hemoglobin gene HvHb1 that oxidizes NO to NO3−. Reduced NO production under drought conditions in UHb plants was associated with increased drought tolerance. Since NO biosynthesis has been related to polyamine metabolism, we investigated whether the observed drought-related NO changes could involve polyamine pathway. UHb plants showed increases in total polyamines and in particular polyamines such as spermidine. These increases correlated with the accumulation of the amino acid precursors of polyamines and with the expression of specific polyamine biosynthesis genes. This suggests a potential interplay between NO and polyamine biosynthesis during drought response. Since ethylene has been linked to NO signaling and it is also related to polyamine metabolism, we explored this connection. In vivo ethylene measurement showed that UHb plants significantly decrease ethylene production and expression of aminocyclopropane-1-carboxylic acid synthase gene, the first committed step in ethylene biosynthesis compared with wild type. These data suggest a NO-ethylene influenced regulatory node in polyamine biosynthesis linked to drought tolerance/susceptibility in barley.

Characterization of the Heme Pocket Structure and Ligand Binding Kinetics of Non-symbiotic Hemoglobins from the Model Legume Lotus japonicus.

Plant hemoglobins (Hbs) are found in nodules of legumes and actinorhizal plants but also in non-symbiotic organs of monocots and dicots. Non-symbiotic Hbs (nsHbs) have been classified into two phylogenetic groups. Class 1 nsHbs show an extremely high O2 affinity and are induced by hypoxia and nitric oxide (NO), whereas class 2 nsHbs have moderate O2 affinity and are induced by cold and cytokinins. The functions of nsHbs are still unclear, but some of them rely on the capacity of hemes to bind diatomic ligands and catalyze the NO dioxygenase (NOD) reaction (oxyferrous Hb + NO → ferric Hb + nitrate). Moreover, NO may nitrosylate Cys residues of proteins. It is therefore important to determine the ligand binding properties of the hemes and the role of Cys residues. Here, we have addressed these issues with the two class 1 nsHbs (LjGlb1-1 and LjGlb1-2) and the single class 2 nsHb (LjGlb2) of Lotus japonicus, which is a model legume used to facilitate the transfer of genetic and biochemical information into crops. We have employed carbon monoxide (CO) as a model ligand and resonance Raman, laser flash photolysis, and stopped-flow spectroscopies to unveil major differences in the heme environments and ligand binding kinetics of the three proteins, which suggest non-redundant functions. In the deoxyferrous state, LjGlb1-1 is partially hexacoordinate, whereas LjGlb1-2 shows complete hexacoordination (behaving like class 2 nsHbs) and LjGlb2 is mostly pentacoordinate (unlike other class 2 nsHbs). LjGlb1-1 binds CO very strongly by stabilizing it through hydrogen bonding, but LjGlb1-2 and LjGlb2 show lower CO stabilization. The changes in CO stabilization would explain the different affinities of the three proteins for gaseous ligands. These affinities are determined by the dissociation rates and follow the order LjGlb1-1 > LjGlb1-2 > LjGlb2. Mutations LjGlb1-1 C78S and LjGlb1-2 C79S caused important alterations in protein dynamics and stability, indicating a structural role of those Cys residues, whereas mutation LjGlb1-1 C8S had a smaller effect. The three proteins and their mutant derivatives exhibited similarly high rates of NO consumption, which were due to NOD activity of the hemes and not to nitrosylation of Cys residues.

Role of Ionic Strength and pH in Modulating Thermodynamic Profiles Associated with CO Escape from Rice Nonsymbiotic Hemoglobin 1.

Type 1 nonsymbiotic hemoglobins are found in a wide variety of land plants and exhibit very high affinities for exogenous gaseous ligands. These proteins are presumed to have a role in protecting plant cells from oxidative stress under etiolated/hypoxic conditions through NO dioxygenase activity. In this study we have employed photoacoustic calorimetry, time-resolved absorption spectroscopy, and classical molecular dynamics simulations in order to elucidate thermodynamics, kinetics, and ligand migration pathways upon CO photodissociation from WT and a H73L mutant of type 1 nonsymbiotic hemoglobin from Oryza sativa (rice). We observe a temperature dependence of the resolved thermodynamic parameters for CO photodissociation from CO-rHb1 which we attribute to temperature dependent formation of a network of electrostatic interactions in the vicinity of the heme propionate groups. We also observe slower ligand escape from the protein matrix under mildly acidic conditions in both the WT and H73L mutant (τ = 134 ± 19 and 90 ± 15 ns). Visualization of transient hydrophobic channels within our classical molecular dynamics trajectories allows us to attribute this phenomenon to a change in the ligand migration pathway which occurs upon protonation of the distal His73, His117, and His152. Protonation of these residues may be relevant to the functioning of the protein in vivo given that etiolation/hypoxia can cause a decrease in intracellular pH in plant cells.