Non-symbiotic Class Research Articles

Overexpression of the spinach S-nitrosoglutathione reductase (SoGSNOR) in tobacco resulted in enhanced nitrate stress tolerance

S-nitrosylation, the addition of nitric oxide (NO) moiety to a reactive cysteine thiol, to form an S-nitrosothiol (SNO), is emerging as a prototypic redox-based post-translational modification. S-nitrosoglutathione reductase (GSNOR) is thought to be the major regulator of total cellular SNO levels in plants. However, its role in excess nitrate stress has not been investigated in spinach. In this study, a spinach GSNOR gene (GenBank Accession No. KR381778) was amplified and designated as SoGSNOR. The transcript and protein level of SoGSNOR were reduced after excess nitrate treatment for 24 h. Addition of NO donor to the nitrate solution decreased the SoGSNOR expression, while supplementation inhibitor of nitrate reductase and nitric oxide synthase increased its expression. Overexpression of SoGSNOR in tobacco increased the germination rate of transgenic seeds, compared to the wild type (WT) under nitrate stress. Higher photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency and expression level of some stress-related genes were detected in the transgenic seedlings than the WT under nitrate stress. The transgenic tobacco seedlings have lower malondialdehyde content, reactive oxygen species (ROS) fluorescence, and higher activities and transcript level of superoxide dismutase, catalase, peroxidase under nitrate stress. SoGSNOR transgenic tobacco plants have lower NR activity and protein level, higher GSNOR and non-symbiotic class 1 hemoglobin (nsHb) protein level than the WT plants, leading to lower NO accumulation and SNOs contents under nitrate stress. These results suggested that overexpression of SoGSNOR increased nitrate stress tolerance of tobacco by regulating ROS and RNS metabolism. A spinach S-nitrosoglutathione reductase (SoGSNOR) gene was isolated from spinach root. The expression level and activities of SoGSNOR were reduced by excess nitrate treatment. Overexpression of SoGSNOR in tobacco increased nitrate stress tolerance by regulating ROS and RNS metabolism.

Expression of the Tobacco Non-symbiotic Class 1 Hemoglobin Gene Hb1 Reduces Cadmium Levels by Modulating Cd Transporter Expression Through Decreasing Nitric Oxide and ROS Level in Arabidopsis.

Hemoglobin (Hb) proteins are ubiquitous in plants, and non-symbiotic class 1 hemoglobin (Hb1) is involved in various biotic and abiotic stress responses. Here, the expression of the tobacco (Nicotiana tabacum) hemoglobin gene NtHb1 in Arabidopsis (Arabidopsis thaliana) showed higher cadmium (Cd) tolerance and lower accumulations of Cd, nitric oxide (NO), and reactive oxygen species (ROS) like hydrogen peroxide (H2O2). NtHb1-expressing Arabidopsis exhibited a reduced induction of NO levels in response to Cd, suggesting scavenging of NO by Hb1. In addition, transgenic plants had reduced accumulation of ROS and increased activities of antioxidative enzymes (catalase, superoxide dismutase, and glutathione reductase) in response to Cd. While the expression of the Cd exporters ABC transporter (PDR8) and Ca2+/H+ exchangers (CAXs) was increased, that of the Cd importers iron responsive transporter 1 (IRT1) and P-type 2B Ca2+ ATPase (ACA10) was reduced in response to Cd. When Col-0 plants were treated with the NO donor sodium nitroprusside (SNP) and H2O2, the expression pattern of Cd transporters (PDR8, CAX3, IRT1, and ACA10) was reversed, suggesting that NtHb1 expression decreased the Cd level by regulating the expression of Cd transporters via decreased NO and ROS. Correspondingly, NtHb1-expressing Arabidopsis showed increased Cd export. In summary, the expression of NtHb1 reduces Cd levels by regulating Cd transporter expression via decreased NO and ROS levels in Arabidopsis.

Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the Lotus japonicus-Mesorhizobium loti symbiosis.

Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia-legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5'-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels.

Over-expression of NtHb1 encoding a non-symbiotic class 1 hemoglobin of tobacco enhances a tolerance to cadmium by decreasing NO (nitric oxide) and Cd levels in Nicotiana tabacum

Tobacco gene NtHb1 encoding a non-symbiotic class 1 hemoglobin was identified to confer cadmium (Cd) tolerance in yeast Saccharomyces cerevisiae by expressing cDNA library of Nicotiana tabacum in WT yeast Y800. To examine the role of NtHb1 in plants, NtHb1 was over-expressed in tobacco (N. tabacum). NtHb1-expressing tobacco exhibited higher tolerance to Cd, but lower levels of Cd compared with vector-expressing control tobacco. Since it was reported that Hb (hemoglobin) is involved in scavenging NO, we investigated NO level in control and NtHb1-expressing tobacco. Interestingly, NO content of NtHb1-tobacco was lower than that of control tobacco in the presence of Cd, while there is no significant difference in NO accumulations of two plants in the absence of Cd. Among Cd transporters, CAX3 (cation/proton exchanger) transcript level of NtHb1-tobacco in response to Cd was higher than control tobacco, but IRT1 (iron-responsive transporter) expression was not. In addition, knockoutArabidopsis of AtNR1 and AtNR2 encoding NO producing enzymes nitrate reductase 1 and nitrate reductase 2 respectively, displayed enhanced Cd tolerance and reduced NO amounts in comparison with control Arabidopsis Col-0. Taken together, it is concluded that the increase of Hb by NtHb1 expression promotes Cd tolerance and reduces Cd accumulation by alleviating the level of NO induced by Cd in transgenic tobacco.

Characterization of haemoglobin from Actinorhizal plants – An in silico approach

Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin.

The responses of Vitreoscilla hemoglobin-expressing hybrid aspen (Populus tremula × tremuloides) exposed to 24-h herbivory: expression of hemoglobin and stress-related genes in exposed and nonorthostichous leaves

The responses of transcriptome and phenolic compounds were determined with Populus tremula L.×Populus tremuloides Michx. expressing the hemoglobin (Hb) of Vitreoscilla (VHb) and non-transformant (wt) line. After 24-h exposure of leaves to Conistra vaccinii L., the transcript levels of endogenous non-symbiotic class 1 Hb (PttHb1) and truncated Hb (PttTrHb) genes were modestly reduced and increased, respectively, in both wt and VHb-expressing line. Besides the herbivory exposed leaves showing the most significant transcriptome changes, alterations were also detected in the transcriptome of nonorthostichous leaves positioned directly above the exposed leaves. Both wt and VHb-expressing line displayed similar herbivory-induced effects on gene expression, although the extent of responses was more pronounced in the wt than in the VHb-expressing line. The contents of phenolic compounds were not altered due to herbivory and they were alike in the wt and VHb-expressing line. In addition, we determined the relative growth rates (RGRs) of Orthosia gothica L., Ectropis crepuscularia Denis & Schiff. and Orgyia antiqua L. larvae, and found no variation in the RGRs between the lines. Thus, VHb-expressing P. tremula×tremuloides lines showed to be comparable with wt in regards to the food quality of leaves.

Respiratory complex I deficiency results in low nitric oxide levels, induction of hemoglobin and upregulation of fermentation pathways

The cytoplasmic male-sterile (CMS) mutant of Nicotiana sylvestris which lacks NAD7, one of the subunits of respiratory complex I (NADH: ubiquinone oxidoreductase, EC 1.6.5.3), is characterized by very low (~10 times lower as compared to the wild type plants) emissions of nitric oxide (NO) under hypoxic conditions. The level of the non-symbiotic class 1 hemoglobin, as shown by Western blotting, is increased compared to the wild type plants not only under hypoxia but this protein reveals its marked expression in the CMS mutant even under normoxic conditions. The activity of aconitase (EC 4.2.1.3) is low in the CMS mutant, especially in the mitochondrial compartment, which indicates the suppression of the tricarboxylic acid cycle. The CMS mutant exhibits the severalfold higher activities of alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27) under the normoxic conditions as compared to the wild type plants. It is concluded that the lack of functional complex I results in upregulation of the pathways of hypoxic metabolism which include both fermentation of pyruvate and scavenging of NO by the non-symbiotic hemoglobin.

Structural and functional properties of class 1 plant hemoglobins

Nonsymbiotic class 1 plant hemoglobins are induced under hypoxia. Structurally they are protein dimers consisting of two identical subunits, each containing heme iron in a weak hexacoordinate state. The weak hexacoordination of heme-iron binding to the distal histidine results in an extremely high avidity to oxygen, with a dissociation constant in the nanomolar range. This low dissociation constant is due to rapid oxygen binding resulting in protein conformational changes that slow dissociation from the heme site. Class 1 hemoglobins are characterized by an increased rate of Fe³(+) reduction which is likely mediated by cysteine residue. This cysteine can form a reversible covalent bond between two monomers as shown by mass spectrometry analysis and, in addition to its structural role, prevents the molecule from autoxidation. The structural properties of class 1 hemoglobins allow them to serve as soluble electron transport proteins in the enzymatic system scavenging nitric oxide produced in low oxygen via reduction of nitrite. During oxygenation of nitric oxide to nitrate, oxidized ferric hemoglobin is formed (methemoglobin), which can be reduced by an associated reductase. The identified candidate for this reduction is monodehydroascorbate reductase. It is suggested that hemoglobin functions as a terminal electron acceptor during the hypoxic turnover of nitrogen, the process aided by its extremely high affinity for oxygen.