Redox Reactivity of Nonsymbiotic Phytoglobins towards Nitrite.

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.

Fine-tuned nitric oxide and hormone interface in plant root development and regeneration.

Plant root growth and developmental capacities reside in a few stem cells of the root apical meristem (RAM). Maintenance of these stem cells requires regenerative divisions of the initial stem cell niche (SCN) cells, self-maintenance, and proliferative divisions of the daughter cells. This ensures sufficient cell diversity to guarantee the development of complex root tissues in the plant. Damage in the root during growth involves the formation of a new post-embryonic root, a process known as regeneration. Post-embryonic root development and organogenesis processes include primary root development and SCN maintenance, plant regeneration, and the development of adventitious and lateral roots. These developmental processes require a fine-tuned balance between cell proliferation and maintenance. An important regulator during root development and regeneration is the gasotransmitter nitric oxide (NO). In this review we have sought to compile how NO regulates cell rate proliferation, cell differentiation, and quiescence of SCNs, usually through interaction with phytohormones, or other molecular mechanisms involved in cellular redox homeostasis. NO exerts a role on molecular components of the auxin and cytokinin signaling pathways in primary roots that affects cell proliferation and maintenance of the RAM. During root regeneration, a peak of auxin and cytokinin triggers specific molecular programs. Moreover, NO participates in adventitious root formation through its interaction with players of the brassinosteroid and cytokinin signaling cascade. Lately, NO has been implicated in root regeneration under hypoxia conditions by regulating stem cell specification through phytoglobins.

Overexpression of a soybean Globin (GmGlb1-1) gene reduces plant susceptibility to Meloidogyne incognita.

MAIN CONCLUSION: The overexpression of the GmGlb1-1 gene reduces plant susceptibility to Meloidogyne incognita. Non-symbiotic globin class #1 (Glb1) genes are expressed in different plant organs, have a high affinity for oxygen, and are related to nitric oxide (NO) turnover. Previous studies showed that soybean Glb1 genes are upregulated in soybean plants under flooding conditions. Herein, the GmGlb1-1 gene was identified in soybean as being upregulated in the nematode-resistant genotype PI595099 compared to the nematode-susceptible cultivar BRS133 during plant parasitism by Meloidogyne incognita. The Arabidopsis thaliana and Nicotiana tabacum transgenic lines overexpressing the GmGlb1-1 gene showed reduced susceptibility to M. incognita. Consistently, gall morphology data indicated that pJ2 nematodes that infected the transgenic lines showed developmental alterations and delayed parasitism progress. Although no significant changes in biomass and seed yield were detected, the transgenic lines showed an elongated, etiolation-like growth under well-irrigation, and also developed more axillary roots under flooding conditions. In addition, transgenic lines showed upregulation of some important genes involved in plant defense response to oxidative stress. In agreement, higher hydrogen peroxide accumulation and reduced activity of reactive oxygen species (ROS) detoxification enzymes were also observed in these transgenic lines. Thus, based on our data and previous studies, it was hypothesized that constitutive overexpression of the GmGlb1-1 gene can interfere in the dynamics of ROS production and NO scavenging, enhancing the acquired systemic acclimation to biotic and abiotic stresses, and improving the cellular homeostasis. Therefore, these collective data suggest that ectopic or nematode-induced overexpression, or enhanced expression of the GmGlb1-1 gene using CRISPR/dCas9 offers great potential for application in commercial soybean cultivars aiming to reduce plant susceptibility to M. incognita.

Nitric oxide function during oxygen deprivation in physiological and stress processes.

Plants are aerobic organisms that have evolved to maintain specific requirements for oxygen (O2), leading to a correct respiratory energy supply during growth and development. There are certain plant developmental cues and biotic or abiotic stress responses where O2 is scarce. This O2 deprivation known as hypoxia may occur in hypoxic niches of plant-specific tissues and during adverse environmental cues such as pathogen attack and flooding. In general, plants respond to hypoxia through a complex reprogramming of their molecular activities with the aim of reducing the impact of stress on their physiological and cellular homeostasis. This review focuses on the fine-tuned regulation of hypoxia triggered by a network of gaseous compounds that includes O2, ethylene, and nitric oxide. In view of recent scientific advances, we summarize the molecular mechanisms mediated by phytoglobins and by the N-degron proteolytic pathway, focusing on embryogenesis, seed imbibition, and germination, and also specific structures, most notably root apical and shoot apical meristems. In addition, those biotic and abiotic stresses that comprise hypoxia are also highlighted.

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.

Hemoglobins in the legume-Rhizobium symbiosis.

Legume nodules have two types of hemoglobins: symbiotic or leghemoglobins (Lbs) and nonsymbiotic or phytoglobins (Glbs). The latter are categorized into three phylogenetic classes differing in heme coordination and O2 affinity. This review is focused on the roles of Lbs and Glbs in the symbiosis of rhizobia with crop legumes and the model legumes for indeterminate (Medicago truncatula) and determinate (Lotus japonicus) nodulation. Only two hemoglobin functions are well established in nodules: Lbs deliver O2 to the bacteroids and act as O2 buffers, preventing nitrogenase inactivation; and Glb1-1 modulates nitric oxide concentration during symbiosis, from the early stage, avoiding the plant's defense response, to nodule senescence. Here, we critically examine early and recent results, update and correct the information on Lbs and Glbs with the latest genome versions, provide novel expression data and identify targets for future research. Crucial unresolved questions include the expression of multiple Lbs in nodules, their presence in the nuclei and in uninfected nodule cells, and, intriguingly, their expression in nonsymbiotic tissues. RNA-sequencing data analysis shows that Lbs are expressed as early as a few hours after inoculation and that their mRNAs are also detectable in roots and pods, which clearly suggests that these heme proteins play additional roles unrelated to nitrogen fixation. Likewise, issues awaiting investigation are the functions of other Glbs in nodules, the spatiotemporal expression profiles of Lbs and Glbs at the mRNA and protein levels, and the molecular mechanisms underlying their regulation during nodule development and in response to stress and hormones.

Plant hemoglobins: a journey from unicellular green algae to vascular plants.

Globins (Glbs) are widely distributed in archaea, bacteria and eukaryotes. They can be classified into proteins with 2/2 or 3/3 α-helical folding around the heme cavity. Both types of Glbs occur in green algae, bryophytes and vascular plants. The Glbs of angiosperms have been more intensively studied, and several protein structures have been solved. They can be hexacoordinate or pentacoordinate, depending on whether a histidine is coordinating or not at the sixth position of the iron atom. The 3/3 Glbs of class 1 and the 2/2 Glbs (also called class 3 in plants) are present in all angiosperms, whereas the 3/3 Glbs of class 2 have been only found in early angiosperms and eudicots. The three Glb classes are expected to play different roles. Class 1 Glbs are involved in hypoxia responses and modulate NO concentration, which may explain their roles in plant morphogenesis, hormone signaling, cell fate determination, nutrient deficiency, nitrogen metabolism and plant-microorganism symbioses. Symbiotic Glbs derive from class 1 or class 2 Glbs and transport O2 in nodules. The physiological roles of class 2 and class 3 Glbs are poorly defined but could involve O2 and NO transport and/or metabolism, respectively. More research is warranted on these intriguing proteins to determine their non-redundant functions.

Nitric oxide and phytoglobin PHYTOGB1 are regulatory elements in the Solanum lycopersicum-Rhizophagus irregularis mycorrhizal symbiosis.

The regulatory role of nitric oxide (NO) and phytoglobins in plant response to pathogenic and mutualistic microbes has been evidenced. However, little is known about their function in the arbuscular mycorrhizal (AM) symbiosis. We investigated whether NO and phytoglobin PHYTOGB1 are regulatory components in the AM symbiosis. Rhizophagus irregularis in vitro-grown cultures and tomato plants were used to monitor AM-associated NO-related root responses as compared to responses triggered by the pathogen Fusarium oxysporum. A genetic approach was conducted to understand the role of PHYTOGB1 on NO signaling during both interactions. After a common early peak in NO levels in response to both fungi, a specific NO accumulation pattern was triggered in tomato roots during the onset of the AM interaction. PHYTOGB1 was upregulated by the AM interaction. By contrast, the pathogen triggered a continuous NO accumulation and a strong downregulation of PHYTOGB1. Manipulation of PHYTOGB1 levels in overexpressing and silenced roots led to a deregulation of NO levels and altered mycorrhization and pathogen infection. We demonstrate that the onset of the AM symbiosis is associated with a specific NO-related signature in the host root. We propose that NO regulation by PHYTOGB1 is a regulatory component of the AM symbiosis.

CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation.

Legume nodules contain high concentrations of leghemoglobins (Lbs) encoded by several genes. The reason for this multiplicity is unknown. CRISPR/Cas9 technology was used to generate stable mutants of the three Lbs of Lotus japonicus. The phenotypes were characterized at the physiological, biochemical and molecular levels. Nodules of the triple mutants were examined by electron microscopy and subjected to RNA-sequencing (RNA-seq) analysis. Complementation studies revealed that Lbs function synergistically to maintain optimal N2 fixation. The nodules of the triple mutants overproduced superoxide radicals and hydrogen peroxide, which was probably linked to activation of NADPH oxidases and changes in superoxide dismutase isoforms expression. The mutant nodules showed major ultrastructural alterations, including vacuolization, accumulation of poly-ß-hydroxybutyrate and disruption of mitochondria. RNA-seq of c. 20 000 genes revealed significant changes in expression of carbon and nitrogen metabolism genes, transcription factors, and proteinases. Lb-deficient nodules had c. 30-50-fold less heme but similar transcript levels of heme biosynthetic genes, suggesting a post-translational regulatory mechanism of heme synthesis. We conclude that Lbs act additively in nodules and that the lack of Lbs results in early nodule senescence. Our observations also provide insight into the reprogramming of the gene expression network associated with Lb deficiency, probably as a result of uncontrolled intracellular free O2 concentration.

Rice phytoglobins regulate responses under low mineral nutrients and abiotic stresses in Arabidopsis thaliana.

Just like animals, plants also contain haemoglobins (known as phytoglobins in plants). Plant phytoglobins (Pgbs) have been categorized into 6 different classes, namely, Phytogb0 (Pgb0), Phytogb1 (Pgb1), Phytogb2 (Pgb2), SymPhytogb (sPgb), Leghaemoglobin (Lb), and Phytogb3 (Pgb3). Among the 6 Phytogbs, sPgb and Lb have been functionally characterized, whereas understanding of the roles of other Pgbs is still evolving. In our present study, we have explored the function of 2 rice Pgbs (OsPgb1.1 and OsPgb1.2). OsPgb1.1, OsPgb1.2, OsPgb1.3, and OsPgb1.4 displayed increased level of transcript upon salt, drought, cold, and ABA treatment. The overexpression (OX) lines of OsPgb1.2 in Arabidopsis showed a tolerant phenotype in terms of better root growth in low potassium (K+ ) conditions. The expression of the known K+ gene markers such as LOX2, HAK5, and CAX3 was much higher in the OsPgb1.2 OX as compared to wild type. Furthermore, the OsPgb1.2 OX lines showed a decrease in reactive oxygen species (ROS) production and conversely an increase in the K+ content, both in root and shoot, as compared to wild type in K+ limiting condition. Our results indicated the potential involvement of OsPgb1.2 in signalling networks triggered by the nutrient deficiency stresses.

Phytoglobin expression influences soil flooding response of corn plants.

Background and Aims Excess water is a limiting factor for crop productivity. Under conditions of full submergence or flooding, plants can experience prolonged oxygen depletion which compromises basic physiological and biochemical processes. Severe perturbations of the photosynthetic machinery with a concomitant decline in photosynthetic potential as a result of elevated levels of reactive oxygen species (ROS) are the major consequences of water excess. Phytoglobins (Pgbs) are ubiquitous proteins induced by several types of stress which affect plant response by modulating nitric oxide. Methods Maize plants overexpressing or downregulating two Pgb genes were subjected to soil flooding for 10 d and their performance was estimated by measuring several gas exchange parameters including photosynthetic rate. Above-ground tissue was utilized to localize ROS and to measure the expression and activities of major antioxidant enzymes. Key Results Relative to the wild type, flooded plants overexpressing Pgb genes retained a greater photosynthetic rate and enhanced activity of several antioxidant enzymes. These plants also exhibited high levels of ascorbic acid and reduced ROS staining. This was in contrast to flooded plants downregulating Pgb genes and characterized by the lowest photosynthetic rates and reduced expression and activities of many antioxidant enzymes. Conclusions Induction of Pgb genes alleviates flooding stress by limiting ROS-induced damage and ensuring a sustained photosynthetic rate. This is achieved through improvements of the ascorbate antioxidant status including an enrichment of the ascorbate pool via de novo and recycling mechanisms, and increased activities of several ROS-scavenging enzymes.

Interplay between the Brassica napus phytoglobin (BnPgb1), folic acid, and antioxidant responses enhances plant tolerance to waterlogging.

Oxygen deprivation by waterlogging reduces the productivity of several crop species, including the oil-producing crop Brassica napus L., which is highly sensitive to excess moisture. Among factors induced by oxygen deficiency are phytoglobins (Pgbs), heme-containing proteins known to ameliorate the response of plants to the stress. This study examined the early responses to waterlogging in B. napus plants over-expressing or down-regulating the class 1 (BnPgb1) and class 2 (BnPgb2) Pgbs. The depression of gas exchange parameters and plant biomass was exacerbated by the suppression of BnPgb1, while suppression of BnPgb2 did not evoke any changes. This suggests that natural occurring levels of BnPgb1 (but not BnPg2) are required for the response of the plants to waterlogging. Typical waterlogging symptoms, including the accumulation of reactive oxygen species (ROS) and the deterioration of the root apical meristem (RAM) were attenuated by over-expression of BnPgb1. These effects were associated with the activation of antioxidant system and the transcriptional induction of folic acid (FA). Pharmacological treatments revealed that high levels of FA were sufficient to revert the inhibitory effect of waterlogging, suggesting that the interplay between BnPgb1, antioxidant responses and FA might contribute to plant tolerance to waterlogging stress.

Transduction of Signals during Somatic Embryogenesis.

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.

Tolerance to excess moisture in soybean is enhanced by over-expression of the Glycine max Phytoglobin (GmPgb1).

Excess moisture in the form of waterlogging or full submergence can cause severe conditions of hypoxia or anoxia compromising several physiological and biochemical processes. A decline in photosynthetic rate due to accumulation of ROS and damage of leaf tissue are the main consequences of excess moisture. These effects compromise crop yield and quality, especially in sensitive species, such as soybean (Glycine max.). Phytoglobins (Pgbs) are expressed during hypoxia and through their ability to scavenge nitric oxide participate in several stress-related responses. Soybean plants over-expressing or suppressing the Pgb1 gene GmPgb1 were generated and their ability to cope with waterlogging and full submergence conditions was assessed. Plants over-expressing GmPgb1 exhibited a higher retention of photosynthetic rate during waterlogging and survival rate during submergence relative to wild type plants. The same plants also had lower levels of ROS due to a reduction in expression of Respiratory Burst Oxidase Homologs (RBOH), components of the NADPH oxidase enzyme, and enhanced antioxidant system characterized by higher expression of catalases (CAT) and superoxide dismutase (SOD), as well as elevated expression and activity of ascorbate peroxidase (APX). Plants over-expressing GmPgb1 also exhibited an expression pattern of aquaporins typical of excess moisture resilience. This was in contrast to plants downregulating GmPgb1 which were characterized by the lowest photosynthetic rates, higher ROS signal, and reduced expression and activities of many antioxidant enzymes. Results from these studies suggest that GmPgb1 exercises a protective role during conditions of excess moisture with similar mechanisms operating during waterlogging and submergence.

Main nitric oxide (NO) hallmarks to relieve arsenic stress in higher plants.

Arsenic (As) is a toxic metalloid that adversely affects plant growth, and poses severe risks to human health. It induces disturbance to many physiological and metabolic pathways such as nutrient, water and redox imbalance, abnormal photosynthesis and ATP synthesis and loss of membrane integrity. Nitric oxide (NO) is a free radical molecule endogenously generated in plant cells which has signalling properties. Under As-stress, the endogenous NO metabolism is significantly affected in a clear connection with the metabolism of reactive oxygen species (ROS) triggering nitro-oxidative stress. However, the exogenous NO application provides beneficial effects under As-stress conditions which can relieve oxidative damages by stimulating the antioxidant systems, regulation of the expression of the transporter and other defence-related genes, modification of root cell wall composition or the biosynthesis of enriched sulfur compounds such phytochelatins (PCs). This review aims to provide up-to-date information on the key NO hallmarks to relieve As-stress in higher plants. Furthermore, it will be analyzed the diverse genetic engineering techniques to increase the endogenous NO content which could open new biotechnological applications, especially in crops under arsenic stress.

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.

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

[This corrects the article DOI: 10.3389/fpls.2020.600336.].

Over-expression of the Zea mays phytoglobin (ZmPgb1.1) alleviates the effect of water stress through shoot-specific mechanisms.

Water deficit limits plant growth and development by interfering with several physiological and molecular processes both in root and shoot tissues. Through their ability to scavenge nitric oxide (NO), phytoglobins (Pgbs) exercise a protective role during several conditions of stress. While their action has been mainly documented in roots, it is unclear whether Pgb exercises a specific and direct role in shoot tissue. We used a Zea mays root-less system to assess how over-expression or down-regulation of ZmPgb1.1 influences the behavior of shoots exposed to polyethylene glycol (PEG)-simulated water deficit. Relative to their WT and ZmPgb1.1 down-regulating counterparts, PEG-treated shoots over-expressing ZmPgb1.1 exhibited a reduced accumulation of ROS and lipid peroxidation. These effects were ascribed to lower transcript levels of Respiratory Burst Oxidase Homolog (RBOH) genes encoding the ROS generating enzyme complex NADPH oxidase, and a more active antioxidant system. Furthermore, over-expression of ZmPgb1.1 attenuated the reduction in osmotic potential and relative water content experienced during water stress, an observation also demonstrated at a whole plant level, possibly through the retention of the expression of three aquaporins involved in water transfer and implicated in drought tolerance. Pharmacological treatments modulating NO and ethylene levels revealed that the ZmPgb1.1 action was mediated by ethylene synthesis and response, with NO acting as an upstream intermediate. Collectively we provide substantial evidence that ZmPgb1.1 exercises a direct role in shoot tissue, independent from that previously reported in roots, which confers tolerance to water stress.