Low-oxygen-induced root bending is altered by phytoglobin1 through mediation of ethylene response factors (ERFs) and auxin signaling.

MAIN CONCLUSION: phytoglobin1 positively regulates root bending in hypoxic Arabidopsis roots through regulation of ethylene response factors and auxin transport. Hypoxia-induced root bending is known to be mediated by the redundant activity of the group VII ethylene response factors (ERFVII) RAP2.12 and HRE2, causing changes in polar auxin transport (PAT). Here, we show that phytoglobin1 (Pgb1), implicated in hypoxic adaptation through scavenging of nitric oxide (NO), can alter root direction under low oxygen. Hypoxia-induced bending is exaggerated in roots over-expressing Pgb1 and attenuated in those where the gene is suppressed. These effects were attributed to Pgb1 repressing both RAP2.12 and HRE2. Expression, immunological and genetic data place Pgb1 upstream of RAP2.12 and HRE2 in the regulation of root bending in oxygen-limiting environments. The attenuation of slanting in Pgb1-suppressing roots was associated with depletion of auxin activity at the root tip because of depression in PAT, while exaggeration of root bending in Pgb1-over-expressing roots with the retention of auxin activity. Changes in PIN2 distribution patterns, suggestive of redirection of auxin movement during hypoxia, might contribute to the differential root bending responses of the transgenic lines. In the end, Pgb1, by regulating NO levels, controls the expression of 2 ERFVIIs which, in a cascade, modulate PAT and, therefore, root bending.

Seed-specific expression of the class 2 Phytoglobin (Pgb2) increases seed oil in Brassica napus.

To examine the function of phytoglobin 2 (Pgb2) on seed oil level in the oil-producing crop Brassica napus L., we generated transgenic plants in which BnPgb2 was over-expressed in the seeds using the cruciferin1 promoter. Over-expression of BnPgb2 elevated the amount of oil, which showed a positive relationship with the level of BnPgb2, without altering the oil nutritional value, as evidenced by the lack of major changes in composition of fatty acids (FA), and key agronomic traits. Two key transcription factors, LEAFY COTYLEDON1 (LEC1) and WRINKLED1 (WRI1), known to promote the synthesis of fatty acids (FA) and potentiate oil accumulation, were induced in BnPgb2 over-expressing seeds. The concomitant induction of several enzymes of sucrose metabolism, SUCROSE SYNTHASE1 (SUS) 1 and 3, FRUCTOSE BISPHOSPHATE ALDOLASE (FPA), and PHOSPHOGLYCERATE KINASE (PGK), and starch synthesis, ADP-GLUCOSE PHOSPHORYLASE (AGPase) suggests that BnPgb2 favors sugar mobilization for FA production. The two plastid FA biosynthetic enzymes SUBUNIT A OF ACETYL-CoA CARBOXYLASE (ACCA2), and MALONYL-CoA:ACP TRANSACYLASE (MCAT) were also up-regulated by the over-expression of BnPgb2. The requirement of BnPgb2 for oil deposition was further evidenced in natural germplasm by the higher levels of BnPgb2 in seeds of high-oil genotypes relative to their low-oil counterparts.

Phytoglobins regulate nitric oxide-dependent abscisic acid synthesis and ethylene-induced program cell death in developing maize somatic embryos.

MAIN CONCLUSION: During maize somatic embryogenesis, suppression of phytoglobins (Pgbs) reduced ABA levels leading to ethylene-induced programmed cell death in the developing embryos. These effects modulate embryonic yield depending on the cellular localization of specific phytoglobin gene expression. Suppression of Zea mays phytoglobins (ZmPgb1.1 or ZmPgb1.2) during somatic embryogenesis induces programmed cell death (PCD) by elevating nitric oxide (NO). While ZmPgb1.1 is expressed in many embryonic domains and its suppression results in embryo abortion, ZmPgb1.2 is expressed in the basal cells anchoring the embryos to the embryogenic tissue. Down-regulation of ZmPgb1.2 is required to induce PCD in these anchor cells allowing the embryos to develop further. Exogenous applications of ABA could reverse the effects caused by the suppression of either of the two ZmPgbs. A depletion of ABA, ascribed to a down-regulation of biosynthetic genes, was observed in those embryonic domains where the respective ZmPgbs were repressed. These effects were mediated by NO. Depletion in ABA content increased the transcription of genes participating in the synthesis and response of ethylene, as well as the accumulation of ethylene, which influenced embryogenesis. Somatic embryo number was reduced by high ethylene levels and increased with pharmacological treatments suppressing ethylene synthesis. The ethylene inhibition of embryogenesis was linked to the production of reactive oxygen species (ROS) and the execution of PCD. Integration of ABA and ethylene in the ZmPgb regulation of embryogenesis is proposed in a model where NO accumulates in ZmPgb-suppressing cells, decreasing the level of ABA. Abscisic acid inhibits ethylene biosynthesis and the NO-mediated depletion of ABA relieves this inhibition causing ethylene to accumulate. Elevated ethylene levels trigger production of ROS and induce PCD. Ethylene-induced PCD in the ZmPgb1.1-suppressing line [ZmPgb1.1 (A)] leads to embryo abortion, while PCD in the ZmPgb1.2-suppressing line [ZmPgb1.2 (A)] results in the elimination of the anchor cells and the successful development of the embryos.

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.

Transcriptional regulation of annexins in Indian Mustard, Brassica juncea and detoxification of ROS in transgenic tobacco plants constitutively expressing AnnBj1.

Annexins in plants constitute a multigene family and there is growing evidence for their involvement in response to various stress treatments. We have cloned and characterized six different annexin genes from mustard. The transcript regulation of these genes was studied in treatments with various signaling molecules, osmotic stress, oxidative stress conditions and wounding. All these annexins were found to be reponsive to Abscisic acid (ABA). Two genes (AnnBj1 and AnnBj3) were found to respond to most of the stress conditions, which suggest their possible role in cross-talk in multiple signaling pathways. Wound response signal methyl jasmonate (mJA) caused rapid and high expression of AnnBj4. We extended our previous study and showed that transgenic tobacco plants heterologously expressing AnnBj1 evidenced mannitol induced ROS detoxification. These results suggest Brassica annexins may have potential role in alleviating abiotic stress, which should be characterized by in vivo function based studies through silencing by RNAi or overexpression in transgenic plants.

Nitrite-driven anaerobic ATP synthesis in barley and rice root mitochondria.

Mitochondria isolated from the roots of barley (Hordeum vulgare L.) and rice (Oryza sativa L.) seedlings were capable of oxidizing external NADH and NADPH anaerobically in the presence of nitrite. The reaction was linked to ATP synthesis and nitric oxide (NO) was a measurable product. The rates of NADH and NADPH oxidation were in the range of 12-16 nmol min(-1) mg(-1) protein for both species. The anaerobic ATP synthesis rate was 7-9 nmol min(-1) mg(-1) protein for barley and 15-17 nmol min(-1) mg(-1) protein for rice. The rates are of the same order of magnitude as glycolytic ATP production during anoxia and about 3-5% of the aerobic mitochondrial ATP synthesis rate. NADH/NADPH oxidation and ATP synthesis were sensitive to the mitochondrial inhibitors myxothiazol, oligomycin, diphenyleneiodonium and insensitive to rotenone and antimycin A. The uncoupler FCCP completely eliminated ATP production. Succinate was also capable of driving ATP synthesis. We conclude that plant mitochondria, under anaerobic conditions, have a capacity to use nitrite as an electron acceptor to oxidize cytosolic NADH/NADPH and generate ATP.

Identification of an intermolecular disulfide bond in barley hemoglobin.

Barley class-1 hemoglobin (Hb) and its mutated version (Cys(79) replaced by Ser) were overexpressed in Escherichia coli and purified to near homogeneity. Nano-electrospray ionization mass spectrometry (nano-ESI MS) showed that the mutated barley Hb was more readily dissociated to a monomer and was more susceptible to denaturation than the native form. The mutated Hb was oxidized to the ferric state approximately 10(3) times faster than the non-mutated form. The increased oxidation of the mutated Hb was a result of substitution of the cysteine with a serine and not a consequence of monomer formation, per se. Tandem mass spectrometry (MS/MS) analysis revealed that Cys(79) participated in intermolecular S-S bond formation. The rates of nitric oxide scavenging by non-mutated and mutated Hb were similar. We conclude that the cysteine residue is an important contributor to the quaternary and tertiary structure of barley hemoglobin. It however has no direct effect on nitric oxide-scavenging activity of barley Hb.

Nitric oxide scavenging by barley hemoglobin is facilitated by a monodehydroascorbate reductase-mediated ascorbate reduction of methemoglobin.

NADH-dependent NO scavenging in barley extracts is linked to hemoglobin (Hb) expression and is inhibited by SH-reagents. Barley Hb has a single cysteine residue. To determine whether this cysteine was critical for NO scavenging, barley Hb and a mutated version, in which the single Cys(79) was replaced by Ser, were over-expressed in Escherichia coli and purified to near homogeneity. The purified proteins exhibited very low NO-scavenging activity (12-14 nmol min(-1) mg(-1) protein) in the presence of NADH or NADPH. This activity was insensitive to SH-reagents. Addition of an extract from barley roots to either of the purified proteins resulted in high NADH-dependent NO turnover in a reaction that was sensitive to SH-reagents. A protein was purified from barley roots and identified by mass-spectrometry analysis as a cytosolic monodehydroascorbate reductase. It efficiently supported NADH-dependent NO scavenging in the presence of either native or mutated barley Hb. Ascorbate strongly facilitated the rate of metHb reduction. The K (m) for Hb was 0.3 microM, for ascorbate 0.6 mM and for NADH 4 microM. The reaction in the presence of monodehydroascorbate reductase was sensitive to SH-reagents with either form of the Hb. We conclude that metHb reduction and NO turnover do not involve direct participation of the Cys(79) residue of barley Hb. NO scavenging is facilitated by monodehydroascorbate reductase mediating a coupled reaction involving ferric Hb reduction in the presence of ascorbate and NADH.

Hemoglobin expression affects ethylene production in maize cell cultures.

The formation of ethylene under different O(2) concentrations and upon addition of nitric oxide (NO) donor, sodium nitroprusside (SNP), was determined using maize (Zea mays L.) cell lines over-expressing (Hb+) or down-regulating (Hb-) hypoxically inducible (class-1) hemoglobin (Hb). Under all treatments, ethylene levels in the Hb- line were 5 to 6.5 times the levels in Hb+ and four to five times the levels in the wild type. Low oxygen partial pressures impaired ethylene formation in maize cell suspension cultures. 1-Amino-cyclopropane-1-carboxylic acid (ACC) oxidase (E.C. 1.14.17.4) mRNA levels did not vary, either between lines or between treatments. There was, however, significantly enhanced ACC oxidase (ACO) activity in the Hb- line relative to the wild type and the Hb+ line. ACO activity in the Hb- line increased under hypoxic conditions and significantly increased upon treatment with NO under normoxic conditions. The results suggest that limiting class-1 hemoglobin protein synthesis increases ethylene formation in maize suspension cells, possibly via the modulation of NO levels.

NO-degradation by alfalfa class 1 hemoglobin (Mhb1): a possible link to PR-1a gene expression in Mhb1-overproducing tobacco plants.

Tobacco plants overproducing alfalfa class 1 hemoglobin (HOT plants) have been shown to have reduced necrotic symptom development. Here, we show that this altered pathogenic response is linked to a significant increase in the nitric oxide (NO)-affected pathogenesis-related (PR-1a) transcript accumulation in the transgenic plants. Homogenates of HOT transgenic seedlings were also found to have higher NO-scavenging activity than non-transformed ones. The NO-scavenging properties of recombinant alfalfa class1 hemoglobin have been examined. Recombinant Mhb1 (rMhb1) was produced in bacteria and purified using polyethylene glycol (10-25%) fractionation, chromatography on DEAE-Sephacel, and Phenyl Superose columns. After the final purification step, the obtained preparations were near homogeneous and had a molecular weight of 44 kDa determined by size-exclusion chromatography and 23 kDa by SDS-PAGE, indicating that rMhb1 is a dimer. The protein participated in NO-degradation activity with NAD(P)H as a cofactor. After ion-exchange columns, addition of FAD was necessary for exhibiting maximal NO-degradation activity. The NAD(P)H-dependent NO-scavenging activity of rMhb1, which is similar to that of barley hemoglobin, supports a conclusion that both monocot and dicot class 1 hemoglobins can affect cellular NO levels by scavenging NO formed during hypoxia, pathogen attack and other stresses.

Purification and characterization of a barley aleurone abscisic acid-binding protein.

A protein designated ABAP1 and encoded by a novel gene (GenBank accession number AF127388) was purified and shown to specifically bind abscisic acid (ABA). ABAP1 protein is a 472-amino acid polypeptide containing a WW protein interaction domain and is induced by ABA in barley aleurone layers. Polyclonal antiidiotypic antibodies (AB2) cross-reacted with purified ABAP1 and with a corresponding 52-kDa protein associated with membrane fractions of ABA-treated barley aleurones. ABAP1 genes were detected in diverse monocot and dicot species, including wheat, tobacco, alfalfa, garden pea, and oilseed rape. The recombinant ABAP1 protein optimally bound (3)H-(+)-ABA at neutral pH. Denatured ABAP1 protein did not bind (3)H-(+)-ABA, nor did bovine serum albumin. The maximum specific binding as shown by Scatchard plot analysis was 0.8 mol of ABA mol(-1) protein with a linear function of r(2) = 0.94, an indication of one ABA-binding site with a dissociation constant (K(d)) of 28 x 10(-9) m. ABA binding in aleurone plasma membranes showed a maximum binding capacity of 330 nmol of ABA g(-1) protein with a K(d) of 26.5 x 10(-9) m. The similarities in the dissociation constants for ABA binding of the recombinant protein and that of the plasma membranes suggest that the protein within the plasma membrane fraction is the native form of ABAP1. The stereospecificity of ABAP1 was established by the incapability of ABA analogs and metabolites, including (-)-ABA, trans-ABA, phaseic acid, dihydrophaseic acid, and (+)-abscisic acid-glucose ester, to displace (3)H-(+)-ABA bound to ABAP1. However, two ABA precursors, (+)-ABA aldehyde and (+)-ABA alcohol, were able to displace (3)H-(+)-ABA, an indication that the structural requirement of ABAP1 at the C-1 position is not strict. Our data show that ABAP1 exerts high binding affinity for ABA. The interaction is reversible, follows saturation kinetics, and has stereospecificity, thus meeting the criteria for an ABA-binding protein.

Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa root cultures under hypoxic stress.

Transgenic alfalfa root cultures expressing sense and antisense barley hemoglobin transcripts were examined under varying levels of atmospheric oxygen. Root cultures overexpressing the hemoglobin gene (Hb+) maintained root growth when placed under 3% oxygen, whereas control cultures or cultures underexpressing hemoglobin (Hb-) experienced 30-70% declines in growth under the same conditions. ATP levels and ATP/ADP ratios for Hb+ lines did not significantly differ in 40 and 3% oxygen, whereas the ATP levels and ATP/ADP ratios in control and Hb- lines were significantly lower under 3% oxygen. Large increases in the production of nitric oxide (NO) were measured in root cultures grown under hypoxic conditions compared to aerobic conditions. The amount of NO accumulated in an Hb- line was 2.5-fold higher than that in the Hb+ line. Treatment of transgenic root lines under 40% oxygen with NO resulted in significant declines in the ATP levels and ATP/ADP ratio of an Hb- line and the control line, with no significant change in an Hb+ line. The root cell structure of an Hb- line showed evidence of cell breakdown under hypoxic growth, whereas an Hb+ line had no evidence of cell breakdown under similar growth conditions. These results lead us to hypothesize that NO is involved in the response of plants to hypoxia and that hemoglobin modulates the levels of NO in the hypoxic cell.

Cigarette smoking and cognitive performance in healthy Swedish adults.

BACKGROUND: The relationship between cigarette smoking and cognitive function was examined in healthy Swedish adults who were participants in the Betula Prospective Cohort Study of Aging, Memory, and Health. SUBJECTS: The data are from those individuals in the Betula study who were self-reported continuous smokers contrasted to those who reported never smoking cigarettes. DESIGN: The dependent variables were cognitive tasks that varied with respect to difficulty and the demand they placed on processing resources. RESULTS: Current smokers performed more poorly than never smokers on the more cognitively demanding tasks; namely, Block Design and free recall. CONCLUSIONS: The findings were interpreted in the light of the assumption that cigarette smoking may exert its greatest deleterious effect on those cognitive tasks that place the heaviest demands on processing resources.