Induction of antibacterial proteins and peptides in the coprophilous mushroom Coprinopsis cinerea in response to bacteria.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αß-defensins (Csαß-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

Structural Insights into the Mode of Action of the Peptide Antibiotic Copsin.

Defensins make up a class of cysteine-rich antimicrobial peptides, expressed by virtually all eukaryotes as part of their innate immune response. Because of their unique mode of action and rapid killing of pathogenic microbes, defensins are considered promising alternatives to clinically applied antibiotics. Copsin is a defensin-like peptide, previously identified in the mushroom Coprinopsis cinerea. It exerts its activity against a range of Gram-positive bacteria by binding to the peptidoglycan precursor lipid II and prevention of proper cell wall formation. In this study, we present a new workflow for the generation, production, and activity-driven selection of copsin derivatives, based on their expression in Pichia pastoris. One hundred fifty-two single-amino acid mutants and combinations thereof allowed the identification of k-copsin, a peptide variant exhibiting significantly enhanced activity against Bacillus subtilis and Staphylococcus aureus. Furthermore, we performed in silico characterizations of membrane interactions of copsin and k-copsin, in the presence and absence of lipid II. The molecular dynamics data highlighted a high variability in lipid II binding, with a preference for the MurNAc moiety with 47 and 35% of the total contacts for copsin and k-copsin, respectively. Mutated amino acids were located in loop regions of k-copsin and shown to be crucial in the perturbation of the bacterial membrane. These structural studies provide a better understanding of how defensins can be developed toward antibacterial therapies less prone to resistance issues.

Dual targeted poplar ferredoxin NADP(+) oxidoreductase interacts with hemoglobin 1.

Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar (Populus tremula × tremuloides) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast (Saccharomyces cerevisiae) Δyhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP(+) oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast Δyhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol.

Copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis.

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/ß-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.

Probing bacterial-fungal interactions at the single cell level.

Interactions between fungi and prokaryotes are abundant in many ecological systems. A wide variety of biomolecules regulate such interactions and many of them have found medicinal or biotechnological applications. However, studying a fungal-bacterial system at a cellular level is technically challenging. New microfluidic devices provided a platform for microscopic studies and for long-term, time-lapse experiments. Application of these novel tools revealed insights into the dynamic interactions between the basidiomycete Coprinopsis cinerea and the bacterium Bacillus subtilis. Direct contact was mediated by polar attachment of bacteria to only a subset of fungal hyphae suggesting a differential competence of fungal hyphae and thus differentiation of hyphae within a mycelium. The fungicidal activity of B. subtilis was monitored at a cellular level and showed a novel mode of action on fungal hyphae.

Evolution, three-dimensional model and localization of truncated hemoglobin PttTrHb of hybrid aspen.

Thus far, research on plant hemoglobins (Hbs) has mainly concentrated on symbiotic and non-symbiotic Hbs, and information on truncated Hbs (TrHbs) is scarce. The aim of this study was to examine the origin, structure and localization of the truncated Hb (PttTrHb) of hybrid aspen (Populus tremula L. × tremuloides Michx.), the model system of tree biology. Additionally, we studied the PttTrHb expression in relation to non-symbiotic class1 Hb gene (PttHb1) using RNAi-silenced hybrid aspen lines. Both the phylogenetic analysis and the three-dimensional (3D) model of PttTrHb supported the view that plant TrHbs evolved vertically from a bacterial TrHb. The 3D model suggested that PttTrHb adopts a 2-on-2 sandwich of α-helices and has a Bacillus subtilis -like ligand-binding pocket in which E11Gln and B10Tyr form hydrogen bonds to a ligand. However, due to differences in tunnel cavity and gate residue (E7Ala), it might not show similar ligand-binding kinetics as in Bs-HbO (E7Thr). The immunolocalization showed that PttTrHb protein was present in roots, stems as well as leaves of in vitro -grown hybrid aspens. In mature organs, PttTrHb was predominantly found in the vascular bundles and specifically at the site of lateral root formation, overlapping consistently with areas of nitric oxide (NO) production in plants. Furthermore, the NO donor sodium nitroprusside treatment increased the amount of PttTrHb in stems. The observed PttTrHb localization suggests that PttTrHb plays a role in the NO metabolism.

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.

The single-domain globin of Vitreoscilla: augmentation of aerobic metabolism for biotechnological applications.

Extensive studies have revealed that large-scale, high-cell density bioreactor cultivations have significant impact on metabolic networks of oxygen-requiring production organisms. Oxygen transfer problems associated with fluid dynamics and inefficient mixing efficiencies result in oxygen gradients, which lead to reduced performance of the bioprocess, decreased product yields, and increased production costs. These problems can be partially alleviated by improving bioreactor configuration and setting, but significant improvements have been achieved by metabolic engineering methods, especially by heterologously expressing Vitreoscilla hemoglobin (VHb). Vast numbers of studies have been accumulating during the past 20 years showing the applicability of VHb to improve growth and product yields in a variety of industrially significant prokaryotic and eukaryotic hosts. The global view on the metabolism of globin-expressing Escherichia coli cells depicts increased energy generation, higher oxygen uptake rates, and a decrease in fermentative by-product excretion. Transcriptome and metabolic flux analysis clearly demonstrate the multidimensional influence of heterologous VHb on the expression of stationary phase-specific genes and on the regulation of cellular metabolic networks. The exact biochemical mechanisms by which VHb is able to improve the oxygen-limited growth remain poorly understood. The suggested mechanisms propose either the delivery of oxygen to the respiratory chain or the detoxification of reactive nitrogen species for the protection of cytochrome activity. The expression of VHb in E. coli bioreactor cultures is likely to assist bacterial growth through providing an increase in available intracellular oxygen, although to fully understand the exact role of VHb in vivo, further analysis will be required.

Reduction of N-linked xylose and fucose by expression of rat beta1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression.

Plant-specific N-glycosylation, such as the introduction of core alpha1,3-fucose and beta1,2-xylose residues, is a major obstacle to the utilization of plant cell- or plant-derived recombinant therapeutic proteins. The beta1,4-N-acetylglucosaminyltransferase III (GnTIII) introduces a bisecting GlcNAc residue into N-glycans, which exerts a high level of substrate mediated control over subsequent modifications, for example inhibiting mammalian core fucosylation. Based on similar findings in plants, we used Nicotianatabacum BY-2 cells to study the effects of localization and expression levels of GnTIII in the remodeling of the plant N-glycosylation pathway. The N-glycans produced by the cells expressing GnTIII were partially bisected and practically devoid of the paucimannosidic type which is typical for N-glycans produced by wildtype BY-2 suspension cultured cells. The proportion of human-compatible N-glycans devoid of fucose and xylose could be increased from an average of 4% on secreted protein from wildtype cells to as high as 59% in cells expressing chimeric GnTIII, named GnTIII(A.th.) replacing its native localization domain with the cytoplasmic tail, transmembrane, and stem region of Arabidopsis thaliana mannosidase II. The changes in N-glycosylation observed were dependent on the catalytic activity of GnTIII, as the expression of catalytically inactive GnTIII mutants did not show a significant effect on N-glycosylation.

Induction of Pseudomonas aeruginosa fhp and fhpR by reactive oxygen species.

In Pseudomonas aeruginosa, flavohemoglobin (Fhp) and its cognate regulator FhpR (PA2665) form a protective regulatory circuit, which responds to reactive nitrogen species and is also capable of protecting cells against nitrosative stress. Recently, it has been shown that the expression of the fhp promoter is regulated not only by FhpR, but also by two new regulators, PA0779 and PA3697. It has also been suggested that the bacterial flavohemoglobins (flavoHbs) could play a crucial role in the protection of cells against reactive oxygen species (ROS). Therefore, the role and function of the Fhp/FhpR system during oxidative stress were studied by assessing the viability and membrane integrity of P. aeruginosa cells and by analyzing the promoter activities of fhp and fhpR upon exposure to paraquat, hydrogen peroxide, and tert-butyl hydroperoxide, under both aerobic and low-oxygen conditions. The results showed that under aerobic conditions, both fhp and fhpR promoters are induced by ROS generated by the stressors. Thus, the Fhp/FhpR system is implicated in the oxidative stress response. ROS-induced fhp promoter activity was dependent on FhpR, PA0779, and PA3697 regulators. Tert-butyl hydroperoxide-induced fhpR promoter activity was found to be highly repressed by PA0779, and FhpR showed negative autoregulation of its own promoter. Under low-oxygen conditions, the activity of the fhp promoter was not inducible by ROS, but fhpR promoter activity was induced by paraquat, and hydrogen peroxide was repressed in both cases by the regulators PA0779 and PA3697.

The production of biopharmaceuticals in plant systems.

Biopharmaceuticals present the fastest growing segment in the pharmaceutical industry, with an ever widening scope of applications. Whole plants as well as contained plant cell culture systems are being explored for their potential as cheap, safe, and scalable production hosts. The first plant-derived biopharmaceuticals have now reached the clinic. Many biopharmaceuticals are glycoproteins; as the Golgi N-glycosylation machinery of plants differs from the mammalian machinery, the N-glycoforms introduced on plant-produced proteins need to be taken into consideration. Potent systems have been developed to change the plant N-glycoforms to a desired or even superior form compared to the native mammalian N-glycoforms. This review describes the current status of biopharmaceutical production in plants for industrial applications. The recent advances and tools which have been utilized to generate glycoengineered plants are also summarized and compared with the relevant mammalian systems whenever applicable.

Intrinsic non-symbiotic and truncated haemoglobins and heterologous Vitreoscilla haemoglobin expression in plants.

To date, haemoglobins (Hbs) have been shown to exist in all kingdoms of life. The least studied and understood groups are plant non-symbiotic haemoglobins (nsHbs) and the recently found plant truncated Hbs (trHbs). From a biotechnological point of view, the best characterized and almost exclusively applied Hb is the bacterial Vitreoscilla haemoglobin (VHb). In this review, the present state of knowledge of structural features and ligand binding kinetics of plant nsHbs and trHbs and their proposed roles as oxygen carriers, oxygen sensors, and for oxygen storage, in nitric oxide (NO) detoxification, and in peroxidase activity are described. Furthermore, in order to predict the functioning of plant Hbs, their characteristics will be compared with those of the better known bacterial globins. In this context, the effects of heterologous applications of VHb on plants are reviewed. Finally, the challenging future of plant Hb research is discussed.

A small-scale method for the preparation of plant N-linked glycans from soluble proteins for analysis by MALDI-TOF mass spectrometry.

The use of plants as production hosts for recombinant glycoproteins, which is rapidly developing, requires methods for fast and reliable analysis of plant N-linked glycans. This study describes a simple small-scale method for the preparation of N-linked glycans from soluble plant protein and analysis thereof by matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Concentration and protease digestion of plant protein as well as deglycosylation is carried out in a single concentrator unit without the need for intermittent purification to minimize adsorptive loss and to facilitate handling. Plant protein is concentrated in a unit with a 5kDa cutoff, and after buffer exchange, pepsin (EC 3.4.23.1) digestion is carried out in the concentrator overnight to obtain peptides as substrates for deglycosylation. Deglycosylation is carried out with peptide-N-glycosidase A (PNGase A; EC 3.5.1.52) for 24h. Released N-glycans are purified using reverse-phase and cation exchange chromatography micro-columns for removal of peptides and desalting. N-Glycans are directly analyzed by MALDI-TOF MS without derivatization. The method for isolation of N-glycans is compatible with secreted proteins from cell culture supernatant as well as with soluble protein extracts from leaf tissue. As little as 5mug of plant glycoprotein is sufficient for N-glycan preparation for MALDI-TOF MS analysis using this method.

Expression of rat beta(1,4)-N-acetylglucosaminyltransferase III in Nicotiana tabacum remodels the plant-specific N-glycosylation.

Plant N-linked glycans differ substantially from their mammalian counterparts, mainly with respect to modifications of the core glycan, which typically contains a beta(1,2)-xylose and an alpha(1,3)-fucose. The addition of a bisecting N-acetylglucosamine residue by beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII) is known to control the processing of N-linked glycans in mammals, for example by preventing alpha(1,6)-fucosylation of the core glycan. In order to outcompete plant-specific beta(1,2)-xylose and alpha(1,3)-fucose modifications, rat GnTIII was expressed either with its native localization domain (GnTIII) or with the cytoplasmic tail, transmembrane domain and stem region (CTS) of Arabidopsis thaliana mannosidase II (ManII) (GnTIII(A.th.)). Both CTSs targeted enhanced yellow fluorescent protein (eYFP) to a brefeldin A-sensitive compartment, indicative of Golgi localization. GnTIII expression increased the fraction of N-glycans devoid of xylose and fucose from 13% +/- 7% in wild-type plants to 60% +/- 8% in plants expressing GnTIII(A.th.). N-Glycans of plants expressing rat GnTIII contained three major glycan structures of complex bisected, complex, or hybrid bisected type, accounting for 70%-85% of the total N-glycans. On expression of GnTIII(A.th.), N-glycans displayed a higher heterogeneity and were of hybrid type. Co-expression of A. thaliana ManII significantly increased the amount of complex bisected structures relative to the plants expressing GnTIII or GnTIII(A.th.), whereas co-expression of human ManII did not redirect the pool of hybrid structures towards complex-type structures. The method described offers the advantage that it can be implemented in any desired plant system for effective removal of beta(1,2)-xylose and alpha(1,3)-fucose from the N-glycan.

Endogenous PttHb1 and PttTrHb, and heterologous Vitreoscilla vhb haemoglobin gene expression in hybrid aspen roots with ectomycorrhizal interaction.

Present knowledge on plant non-symbiotic class-1 (Hb1) and truncated (TrHb) haemoglobin genes is almost entirely based on herbaceous species while the corresponding tree haemoglobin genes are not well known. The function of these genes has recently been linked with endosymbioses between plants and microbes. In this work, the coding sequences of hybrid aspen (Populus tremulaxtremuloides) PttHb1 and PttTrHb were characterized, indicating that the key residues of haem and ligand binding of both genes were conserved in the deduced amino acid sequences. The expression of PttHb1 and PttTrHb was examined in parallel with that of the heterologous Vitreoscilla haemoglobin gene (vhb) during ectomycorrhiza/ectomycorrhizal (ECM) interaction. Both ECM fungi studied, Leccinum populinum and Xerocomus subtomentosus, enhanced root formation and subsequent growth of roots of all hybrid aspen lines, but only L. populinum was able to form mycorrhizas. Real-time PCR results show that the dual culture with the ECM fungus, with or without emergence of symbiotic structures, increased the expression of both PttHb1 and PttTrHb in the roots of non-transgenic hybrid aspens. PttHb1 and PttTrHb had expression peaks 5 h and 2 d after inoculation, respectively, pointing to different functions for these genes during interaction with root growth-improving fungi. In contrast, ECM fungi were not able to enhance the expression of hybrid aspen endogenous haemoglobin genes in the VHb lines, which may be a consequence of the compensating action of heterologous haemoglobin.

Assessment of biotechnologically relevant characteristics of heterologous hemoglobins in E. coli.

The use of the heterologous bacterial hemoglobin (VHb) from Vitreoscilla to enhance growth and productivity of Escherichia coli under conditions of oxygen limitation has been one of the foremost examples of metabolic engineering. Although VHb has earned its merits during the last two decades by providing enhanced physiological enhancements to organisms from all kingdoms of life, it has been the candidate of choice primarily for historical reasons. Findings made during the last years, however, suggest that hemoglobin and flavohemoglobin proteins from bacterial species other than Vitreoscilla or artificially generated mutant proteins or fusion variants of hemoglobins and flavohemoglobins may be better suited for use in biotechnological processes. This account provides guidelines for the assessment of biotechnologically relevant characteristics conferred by such novel heterologous hemoglobins and flavohemoglobins in E. coli.

Transcriptional activity of Pseudomonas aeruginosa fhp promoter is dependent on two regulators in addition to FhpR.

The regulation of flavohemoglobin expression is complex and depending on its host organism requires a wide variety of different transcriptional regulators. In Pseudomonas aeruginosa, the flavohemoglobin (Fhp) and its cognate regulator FhpR form an NO-sensing and detoxifying system regulated by their common bidirectional promoter Pfhp/PfhpR. The intergenic fhp-fhpR region of P. aeruginosa PAO1 was used as a bait to isolate proteins affecting the transcription of fhp and fhpR. In addition to the FhpR, we identified two previously uncharacterized P. aeruginosa proteins, PA0779 and PA3697. Both PA0779 and PA3697 were found to be essential for NO3(-) and NO2(-) induced Pfhp activity under aerobic and low-oxygen conditions, and needed for the full function of Pfhp/PfhpR as NO responsive regulatory circuit under aerobic conditions. In addition, we show that the transcriptional activity of PfhpR is highly inducible upon addition of SNP under aerobic conditions, but not by NO3(-), NO2(-) or under low-oxygen conditions, supporting the findings that FhpR is not the only factor affecting flavohemoglobin expression in P. aeruginosa.

Impact of the small RNA RyhB on growth, physiology and heterologous protein expression in Escherichia coli.

The small noncoding RNA RyhB is a regulator of iron homeostasis in Escherichia coli. During iron limitation, it downregulates the expression of a number of iron-containing proteins, including enzymes of the tricarboxylic acid cycle and the respiratory chain. Because this infers a potential for RyhB to limit energy metabolism and biosynthetic capacity, the effect of knocking out ryhB on the physiology and heterologous protein productivity of E. coli has been analyzed. During iron limitation, induced either through insufficient extracellular supply or through overexpression of an iron-containing protein, ryhB mutants showed unaltered growth and substrate consumption. They did, however, exhibit significantly lowered acetate production rates. Plasmid-based expression of green fluorescent protein and the heterologous Vitreoscilla hemoglobin VHb was negatively affected by the ryhB knock-out.

Analysis of the contribution of the globin and reductase domains to the ligand-binding properties of bacterial haemoglobins.

Bacterial Hbs (haemoglobins), like VHb (Vitreoscilla sp. Hb), and flavoHbs (flavohaemoglobins), such as FHP (Ralstonia eutropha flavoHb), have different autoxidation and ligand-binding rates. To determine the influence of each domain of flavoHbs on ligand binding, we have studied the kinetic ligand-binding properties of oxygen, carbon monoxide and nitric oxide to the chimaeric proteins, FHPg (truncated form of FHP comprising the globin domain alone) and VHb-Red (fusion protein between VHb and the C-terminal reductase domain of FHP) and compared them with those of their natural counterparts, FHP and VHb. Moreover, we also analysed polarity and solvent accessibility to the haem pocket of these proteins. The rate constants for the engineered proteins, VHb-Red and FHPg, do not differ significantly from those of their natural counterparts, VHb and FHP respectively. Our results suggest that the globin domain structure controls the reactivity towards oxygen, carbon monoxide and nitric oxide. The presence or absence of a reductase domain does not affect the affinity to these ligands.

Heterologous expression of Vitreoscilla haemoglobin in barley (Hordeum vulgare).

The vhb gene encoding Vitreoscilla haemoglobin (VHb) was transferred to barley with the aim of studying the role of oxygen availability in germination and growth. Previous findings indicate that VHb expression improves the efficiency of energy generation during oxygen-limited growth, and germination is known to be an energy demanding growth stage during which the embryos also suffer from oxygen deficiency. When subjected to oxygen deficiency, the roots of vhb-expressing barley plants showed a smaller increase in alcohol dehydrogenase (ADH) activity than those of the control plants. This indicates that VHb plants experienced less severe oxygen deficiency than the control plants, possibly due to the ability of VHb to substitute ADH for recycling NADH and maintaining glycolysis. In contrast to previous findings, we found that constitutive vhb expression did not improve the germination rate of barley kernels in any of the conditions studied. In some cases, vhb expression even slowed down germination slightly. VHb production also appeared to restrict root formation in young seedlings. The adverse effects of VHb on germination and root growth may be related to its ability to scavenge nitric oxide (NO), an important signal molecule in both seed germination and root formation. Because NO has both cytotoxic and stimulating properties, the effect of vhb expression in plants may depend on the level and role of endogenous NO in the conditions studied. VHb production also affected the levels of endogenous barley haemoglobin, which may explain the relatively moderate effects of VHb in this study.