Search Results (15)

Nonsymbiotic phytoglobins (nsHbs) are a diverse superfamily of hemoproteins grouped into three different classes (1, 2, and 3) based on their sequences. Class 1 Hb are expressed under hypoxia, osmotic stress, and/or nitric oxide exposure, while class 2 Hb are induced by cold stress and cytokinins. Both are mainly six-coordinated. The deoxygenated forms of the class 1 and 2 nsHbs from A. thaliana (AtHb1 and AtHb2) are able to reduce nitrite to nitric oxide via a mechanism analogous to other known globins. NsHbs provide a viable pH-dependent pathway for NO generation during severe hypoxia via nitrite reductase-like activity with higher rate constants compared to mammalian globins. These high kinetic parameters, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant hemoglobins could indeed serve as anaerobic nitrite reductases in vivo. The third class of nsHb, also known as truncated hemoglobins, have a compact 2/2 structure and are pentacoordinated, and their exact physiological role remains mostly unknown. To date, no reports are available on the nitrite reductase activity of the truncated AtHb3. In the present work, three representative nsHbs of the plant model Arabidopsis thaliana are presented, and their nitrite reductase-like activity and involvement in nitrosative stress is discussed. The reaction kinetics and mechanism of nitrite reduction by nsHbs (deoxy and oxy form) at different pHs were studied by means of UV-Vis spectrophotometry, along with EPR spectroscopy. The reduction of nitrite requires an electron supply, and it is favored in acidic conditions. This reaction is critically affected by molecular oxygen, since oxyAtHb will catalyze nitric oxide deoxygenation. The process displays unique autocatalytic kinetics with metAtHb and nitrate as end-products for AtHb1 and AtHb2 but not for the truncated one, in contrast with mammalian globins. Full article

Gaseous factors affect post-harvest physiological processes in horticultural crops, including ornamental flowers. However, the molecular responses of cut flowers to the low-oxygen conditions associated with modified atmosphere packaging (MAP) have not yet been elucidated. Here, we show that storage of cut carnation flowers in a sealed polypropylene bag decreased the oxygen concentration in the bag to 3–5% and slowed flower opening. The vase life of carnation flowers after storage for seven days under MAP conditions was comparable to that without storage and was improved by the application of a commercial-quality preservative. The adenylate energy charge (AEC) was maintained at high levels in petals from florets stored under MAP conditions. This was accompanied by the upregulation of four hypoxia-related genes, among which the HYPOXIA-RESPONSIVE ETHYLENE RESPONSE FACTOR and PHYTOGLOBIN genes (DcERF19 and DcPGB1) were newly identified. These results suggest that hypoxia-responsive genes contribute to the maintenance of the energy status in carnation flowers stored under MAP conditions, making this gas-controlling technique potentially effective for maintaining cut flower quality without cooling. Full article

Plants continuously interact with fungi, some of which, such as Fusarium oxysporum, are lethal, leading to reduced crop yields. Recently, nitric oxide (NO) has been found to play a regulatory role in plant responses to F. oxysporum, although the underlying mechanisms involved are poorly understood. In this study, we show that Arabidopsis mutants with altered levels of phytoglobin 1 (Glb1) have a higher survival rate than wild type (WT) after infection with F. oxysporum, although all the genotypes analyzed exhibited a similar fungal burden. None of the defense responses that were analyzed in Glb1 lines, such as phenols, iron metabolism, peroxidase activity, or reactive oxygen species (ROS) production, appear to explain their higher survival rates. However, the early induction of the PR genes may be one of the reasons for the observed survival rate of Glb1 lines infected with F. oxysporum. Furthermore, while PR1 expression was induced in Glb1 lines very early on the response to F. oxysporum, this induction was not observed in WT plants. Full article

Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled (²H, ¹³C and ¹⁵N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the ¹H-¹⁵N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins’ roles in planta. Full article

Drought is an environmental stress that strongly impacts plants. It affects all stages of growth and induces profound disturbances that influence all cellular functions. Legumes can establish a symbiosis with Rhizobium-type bacteria, whose function is to fix atmospheric nitrogen in organs called nodules and to meet plant nitrogen needs. Symbiotic nitrogen fixation (SNF) is particularly sensitive to drought. We raised the hypothesis that, in drought-stressed nodules, SNF inhibition is partly correlated to hypoxia resulting from nodule structure compaction and an increased O₂ diffusion barrier, and that the nodule energy regeneration involves phytoglobin–nitric oxide (Pgb–NO) respiration. To test this hypothesis, we subjected faba bean (Vicia faba L.) plants nodulated with a Rhizobium laguerreae strain to either drought or osmotic stress. We monitored the N₂-fixation activity, the energy state (ATP/ADP ratio), the expression of hypoxia marker genes (alcohol dehydrogenase and alanine aminotransferase), and the functioning of the Pgb–NO respiration in the nodules. The collected data confirmed our hypothesis and showed that (1) drought-stressed nodules were subject to more intense hypoxia than control nodules and (2) NO production increased and contributed via Pgb–NO respiration to the maintenance of the energy state of drought-stressed nodules. Full article

Oxygen (O₂) is the most crucial substrate for numerous biochemical processes in plants. Its deprivation is a critical factor that affects plant growth and may lead to death if it lasts for a long time. However, various biotic and abiotic factors cause O₂ deprivation, leading to hypoxia and anoxia in plant tissues. To survive under hypoxia and/or anoxia, plants deploy various mechanisms such as fermentation paths, reactive oxygen species (ROS), reactive nitrogen species (RNS), antioxidant enzymes, aerenchyma, and adventitious root formation, while nitrate (NO₃⁻), nitrite (NO₂⁻), and nitric oxide (NO) have shown numerous beneficial roles through modulating these mechanisms. Therefore, in this review, we highlight the role of reductive pathways of NO formation which lessen the deleterious effects of oxidative damages and increase the adaptation capacity of plants during hypoxia and anoxia. Meanwhile, the overproduction of NO through reductive pathways during hypoxia and anoxia leads to cellular dysfunction and cell death. Thus, its scavenging or inhibition is equally important for plant survival. As plants are also reported to produce a potent greenhouse gas nitrous oxide (N₂O) when supplied with NO₃⁻ and NO₂⁻, resembling bacterial denitrification, its role during hypoxia and anoxia tolerance is discussed here. We point out that NO reduction to N₂O along with the phytoglobin-NO cycle could be the most important NO-scavenging mechanism that would reduce nitro-oxidative stress, thus enhancing plants’ survival during O₂-limited conditions. Hence, understanding the molecular mechanisms involved in reducing NO toxicity would not only provide insight into its role in plant physiology, but also address the uncertainties seen in the global N₂O budget. Full article

Phytoglobins (Pgbs) are plant-originating heme proteins of the globin superfamily with varying degrees of hexacoordination. Pgbs have a conserved cysteine residue, the role of which is poorly understood. In this paper, we investigated the functional and structural role of cysteine in BvPgb1.2, a Class 1 Pgb from sugar beet (Beta vulgaris), by constructing an alanine-substituted mutant (Cys86Ala). The substitution had little impact on structure, dimerization, and heme loss as determined by X-ray crystallography, size-exclusion chromatography, and an apomyoglobin-based heme-loss assay, respectively. The substitution significantly affected other important biochemical properties. The autoxidation rate increased 16.7- and 14.4-fold for the mutant versus the native protein at 25 °C and 37 °C, respectively. Thermal stability similarly increased for the mutant by ~2.5 °C as measured by nano-differential scanning fluorimetry. Monitoring peroxidase activity over 7 days showed a 60% activity decrease in the native protein, from 33.7 to 20.2 U/mg protein. When comparing the two proteins, the mutant displayed a remarkable enzymatic stability as activity remained relatively constant throughout, albeit at a lower level, ~12 U/mg protein. This suggests that cysteine plays an important role in BvPgb1.2 function and stability, despite having seemingly little effect on its tertiary and quaternary structure. Full article

Soybean (Glycine max) is an economically important crop which is very susceptible to salt stress. Tolerance to Na₂SO₄ stress was evaluated in soybean plants overexpressing or suppressing the phytoglobin GmPgb1. Salt stress depressed several gas exchange parameters, including the photosynthetic rate, caused leaf damage, and reduced the water content and dry weights. Lower expression of respiratory burst oxidase homologs (RBOHB and D), as well as enhanced antioxidant activity, resulting from GmPgb1 overexpression, limited ROS-induced damage in salt-stressed leaf tissue. The leaves also exhibited higher activities of the H₂O₂-quenching enzymes, catalase (CAT) and ascorbate peroxidase (APX), as well as enhanced levels of ascorbic acid. Relative to WT and GmPgb1-suppressing plants, overexpression of GmPgb1 attenuated the accumulation of foliar Na⁺ and exhibited a lower Na⁺/K⁺ ratio. These changes were attributed to the induction of the Na⁺ efflux transporter SALT OVERLY SENSITIVE 1 (SOS1) limiting Na⁺ intake and transport and the inward rectifying K⁺ channel POTASSIUM TRANSPORTER 1 (AKT1) required for the maintenance of the Na⁺/K⁺ balance. Full article

Barley (Hordeum vulgare L.) is the most susceptible cereal species to excess moisture stress. Waterlogging-induced hypoxia causes major morphological, physiological, and metabolic changes, some of which are regulated by the action of plant growth regulators and signal molecules including nitric oxide. Recent studies have evidenced the participation of phytoglobins in attenuating hypoxic stress during conditions of excessive moisture through their ability to scavenge nitric oxide and influence the synthesis and response of growth regulators. This review will highlight major cellular changes linked to plant responses to waterlogging stress with emphasis on phytoglobins. Full article

Somatic embryogenesis (SE) is an in vitro biological process in which bipolar structures (somatic embryos) can be induced to form from somatic cells and regenerate into whole plants. Acquisition of the embryogenic potential in culture is initiated when some competent cells within the explants respond to inductive signals (mostly plant growth regulators, PRGs), and de-differentiate into embryogenic cells. Such cells, “canalized” into the embryogenic developmental pathway, are able to generate embryos comparable in structure and physiology to their in vivo counterparts. Genomic and transcriptomic studies have identified several pathways governing the initial stages of the embryogenic process. In this review, the authors emphasize the importance of the developmental signals required for the progression of embryo development, starting with the de-differentiation of somatic cells and culminating with tissue patterning during the formation of the embryo body. The action and interaction of PGRs are highlighted, along with the participation of master regulators, mostly transcription factors (TFs), and proteins involved in stress responses and the signal transduction required for the initiation of the embryogenic process. Full article

Salinity stress is a major threat to agriculture and global food security. Chemical priming is a promising approach to improving salinity stress tolerance in plants. To identify small molecules with the capacity to enhance salinity stress tolerance in plants, chemical screening was performed using Arabidopsis thaliana. We screened 6400 compounds from the Nagoya University Institute of Transformative Bio-Molecule (ITbM) chemical library and identified one compound, Natolen128, that enhanced salinity-stress tolerance. Furthermore, we isolated a negative compound of Natolen128, namely Necolen124, that did not enhance salinity stress tolerance, though it has a similar chemical structure to Natolen128. We conducted a transcriptomic analysis of Natolen128 and Necolen124 to investigate how Natolen128 enhances high-salinity stress tolerance. Our data indicated that the expression levels of 330 genes were upregulated by Natolen128 treatment compared with that of Necolen124. Treatment with Natolen128 increased expression of hypoxia-responsive genes including ethylene biosynthetic enzymes and PHYTOGLOBIN, which modulate accumulation of nitric oxide (NO) level. NO was slightly increased in plants treated with Natolen128. These results suggest that Natolen128 may regulate NO accumulation and thus, improve salinity stress tolerance in A. thaliana. Full article

The increasing occurrence of floods hinders agricultural crop production and threatens global food security. The majority of vegetable crops are highly sensitive to flooding and it is unclear how these plants use flooding signals to acclimate to impending oxygen deprivation (hypoxia). Previous research has shown that the early flooding signal ethylene augments hypoxia responses and improves survival in Arabidopsis. To unravel how cultivated and wild Solanum species integrate ethylene signaling to control subsequent hypoxia acclimation, we studied the transcript levels of a selection of marker genes, whose upregulation is indicative of ethylene-mediated hypoxia acclimation in Arabidopsis. Our results suggest that ethylene-mediated hypoxia acclimation is conserved in both shoots and roots of the wild Solanum species bittersweet (Solanum dulcamara) and a waterlogging-tolerant potato (Solanum tuberosum) cultivar. However, ethylene did not enhance the transcriptional hypoxia response in roots of a waterlogging-sensitive potato cultivar, suggesting that waterlogging tolerance in potato could depend on ethylene-controlled hypoxia responses in the roots. Finally, we show that ethylene rarely enhances hypoxia-adaptive genes and does not improve hypoxia survival in tomato (Solanum lycopersicum). We conclude that analyzing genes indicative of ethylene-mediated hypoxia acclimation is a promising approach to identifying key signaling cascades that confer flooding tolerance in crops. Full article

To understand how the class 1 phytoglobin is involved in germination process via the modulation of the nitric oxide (NO) metabolism, we performed the analysis of physiological and molecular parameters in the embryos of transgenic barley (Hordeum vulgare L. cv Golden Promise) plants differing in expression levels of the phytoglobin (Pgb1) gene during the first 48 h of germination. Overexpression of Pgb1 resulted in a higher rate of germination, higher protein content and higher ATP/ADP ratios. This was accompanied by a lower rate of NO emission after radicle protrusion, as compared to the wild type and downregulating line, and a lower rate of S-nitrosylation of proteins in the first hours postimbibition. The rate of fermentation estimated by the expression and activity of alcohol dehydrogenase was significantly higher in the Pgb1 downregulating line, the same tendency was observed for nitrate reductase expression. The genes encoding succinate dehydrogenase and pyruvate dehydrogenase complex subunits were more actively expressed in embryos of the seeds overexpressing Pgb1. It is concluded that Pgb1 expression in embryo is essential for the maintenance of redox and energy balance before radicle protrusion, when seeds experience low internal oxygen concentration and exerts the effect on metabolism during the initial development of seedlings. Full article

Overexpression of phytoglobins (formerly plant hemoglobins) increases the survival rate of plant tissues under hypoxia stress by the following two known mechanisms: (1) scavenging of nitric oxide (NO) in the phytoglobin/NO cycle and (2) mimicking ethylene priming to hypoxia when NO scavenging activates transcription factors that are regulated by levels of NO and O₂ in the N-end rule pathway. To map the cellular and metabolic effects of hypoxia in barley (Hordeum vulgare L., cv. Golden Promise), with or without priming to hypoxia, we studied the proteome and metabolome of wild type (WT) and hemoglobin overexpressing (HO) plants in normoxia and after 24 h hypoxia (WT24, HO24). The WT plants were more susceptible to hypoxia than HO plants. The chlorophyll a + b content was lowered by 50% and biomass by 30% in WT24 compared to WT, while HO plants were unaffected. We observed an increase in ROS production during hypoxia treatment in WT seedlings that was not observed in HO seedlings. We identified and quantified 9694 proteins out of which 1107 changed significantly in abundance. Many proteins, such as ion transporters, Ca²⁺-signal transduction, and proteins related to protein degradation were downregulated in HO plants during hypoxia, but not in WT plants. Changes in the levels of histones indicates that chromatin restructuring plays a role in the priming of hypoxia. We also identified and quantified 1470 metabolites, of which the abundance of >500 changed significantly. In summary the data confirm known mechanisms of hypoxia priming by ethylene priming and N-end rule activation; however, the data also indicate the existence of other mechanisms for hypoxia priming in plants. Full article

Flooding limits biomass production in agriculture. Leguminous plants, important agricultural crops, use atmospheric dinitrogen gas as nitrogen nutrition by symbiotic nitrogen fixation with rhizobia, but this root-nodule symbiosis is sometimes broken down by flooding of the root system. In this study, we analyzed the effect of flooding on the symbiotic system of transgenic Lotus japonicus lines which overexpressed class 1 phytoglobin (Glb1) of L. japonicus (LjGlb1-1) or ectopically expressed that of Alnus firma (AfGlb1). In the roots of wild-type plants, flooding increased nitric oxide (NO) level and expression of senescence-related genes and decreased nitrogenase activity; in the roots of transgenic lines, these effects were absent or less pronounced. The decrease of chlorophyll content in leaves and the increase of reactive oxygen species (ROS) in roots and leaves caused by flooding were also suppressed in these lines. These results suggest that increased levels of Glb1 help maintain nodule symbiosis under flooding by scavenging NO and controlling ROS. Full article