Metabolic consequences of knocking out UGT85B1, the gene encoding the glucosyltransferase required for synthesis of dhurrin in Sorghum bicolor (L. Moench).

Many important food crops produce cyanogenic glucosides as natural defense compounds to protect against herbivory or pathogen attack. It has also been suggested that these nitrogen-based secondary metabolites act as storage reserves of nitrogen. In sorghum, three key genes, CYP79A1, CYP71E1 and UGT85B1, encode two Cytochrome P450s and a glycosyltransferase, respectively, the enzymes essential for synthesis of the cyanogenic glucoside dhurrin. Here, we report the use of targeted induced local lesions in genomes (TILLING) to identify a line with a mutation resulting in a premature stop codon in the N-terminal region of UGT85B1. Plants homozygous for this mutation do not produce dhurrin and are designated tcd2 (totally cyanide deficient 2) mutants. They have reduced vigor, being dwarfed, with poor root development and low fertility. Analysis using liquid chromatography-mass spectrometry (LC-MS) shows that tcd2 mutants accumulate numerous dhurrin pathway-derived metabolites, some of which are similar to those observed in transgenic Arabidopsis expressing the CYP79A1 and CYP71E1 genes. Our results demonstrate that UGT85B1 is essential for formation of dhurrin in sorghum with no co-expressed endogenous UDP-glucosyltransferases able to replace it. The tcd2 mutant suffers from self-intoxication because sorghum does not have a feedback mechanism to inhibit the initial steps of dhurrin biosynthesis when the glucosyltransferase activity required to complete the synthesis of dhurrin is lacking. The LC-MS analyses also revealed the presence of metabolites in the tcd2 mutant which have been suggested to be derived from dhurrin via endogenous pathways for nitrogen recovery, thus indicating which enzymes may be involved in such pathways.

Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis.

Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

A combined biochemical screen and TILLING approach identifies mutations in Sorghum bicolor L. Moench resulting in acyanogenic forage production.

Cyanogenic glucosides are present in several crop plants and can pose a significant problem for human and animal consumption, because of their ability to release toxic hydrogen cyanide. Sorghum bicolor L. contains the cyanogenic glucoside dhurrin. A qualitative biochemical screen of the M2 population derived from EMS treatment of sorghum seeds, followed by the reverse genetic technique of Targeted Induced Local Lesions in Genomes (TILLING), was employed to identify mutants with altered hydrogen cyanide potential (HCNp). Characterization of these plants identified mutations affecting the function or expression of dhurrin biosynthesis enzymes, and the ability of plants to catabolise dhurrin. The main focus in this study is on acyanogenic or low cyanide releasing lines that contain mutations in CYP79A1, the cytochrome P450 enzyme catalysing the first committed step in dhurrin synthesis. Molecular modelling supports the measured effects on CYP79A1 activity in the mutant lines. Plants harbouring a P414L mutation in CYP79A1 are acyanogenic when homozygous for this mutation and are phenotypically normal, except for slightly slower growth at early seedling stage. Detailed biochemical analyses demonstrate that the enzyme is present in wild-type amounts but is catalytically inactive. Additional mutants capable of producing dhurrin at normal levels in young seedlings but with negligible leaf dhurrin levels in mature plants were also identified. No mutations were detected in the coding sequence of dhurrin biosynthetic genes in this second group of mutants, which are as tall or taller, and leafier than nonmutated lines. These sorghum mutants with reduced or negligible dhurrin content may be ideally suited for forage production.

Use of the wound-inducible NtQPT2 promoter from Nicotiana tabacum for production of a plant-made vaccine.

The wound-inducible quinolinate phosphoribosyl transferase promoter from Nicotiana tabacum (NtQPT2) was assessed for its capacity to produce B-subunit of the heat-labile toxin (LTB) from enterotoxigenic Escherichia coli in transgenic plant tissues. Comparisons were made with the widely used and constitutive Cauliflower Mosaic Virus 35S (CaMV35S) promoter. The NtQPT2 promoter produced somewhat lower average concentrations of LTB protein per unit weight of hairy root tissue but allowed better growth thereby producing similar or higher overall average yields of LTB per culture batch. Transgenic tobacco plants containing the NtQPT2-LTB construct contained LTB protein in roots but not leaves. Moreover, wounding NtQPT2-LTB transgenic plants, by removal of apices, resulted in an approximate 500% increase in LTB levels in roots when analysed several days later. CaMV35S-LTB transgenic plants contained LTB protein in leaves and roots but wounding made no difference to their LTB content.

Hairy roots cultures from different Solanaceous species have varying capacities to produce E. coli B-subunit heat-labile toxin antigen.

The gene encoding enterotoxigenic Escherichia coli B-subunit heat-labile toxin (LTB) antigen was co-transformed into hairy root cultures of Nicotiana tabacum (tobacco), Solanum lycopersicum (tomato) and Petunia parodii (petunia) under the CaMV35S promoter. Tobacco and petunia roots contained ~65-70 µg LTB g(-1) tissue whilst hairy roots of tomato contained ~10 µg LTB g(-1). Antigen at ~600 ng ml(-1) was detected in growth medium of tobacco and petunia. Tobacco roots with higher LTB levels showed growth retardation of ~80% whereas petunia hairy roots with similar levels of LTB showed only ~35% growth retardation, relative to vector controls. Regeneration of plants from LTB-containing tobacco hairy roots was readily achieved and re-initiated hairy roots from greenhouse-grown plants showed similar growth and LTB production characteristics as the original hairy root cultures.

Temporal strength changes from resistance exercise and albuterol on unloaded muscle.

To assess unloaded knee extensor temporal strength changes, healthy subjects without asthma performed 40 continuous days of unilateral limb suspension, whereby their left leg refrained from normal weight-bearing and ambulatory activity. During the 40-day period, subjects performed resistance exercise (REX) with their unloaded leg on an inertial resistance ergometer and, as part of a double-blind design, consumed the maximal oral therapeutic dosage of albuterol (i.e., 16 mg.d) or a placebo (i.e., lactose) with no crossover. Workout data were partitioned into 4 10-day periods that ran consecutively. Dependent strength variables included concentric total work, eccentric total work, concentric average power (CAP), and eccentric average power (EAP). Dependent variables were analyzed with 5 (time) x 2 (group) x 2 (gender) mixed factorial analyses of variance and the Tukey honestly significant difference test. Concentric total work, CAP, and EAP each demonstrated a time-group-gender (p < 0.05) interaction. Female REX-placebo subjects had the greatest percentage of unloaded knee extensor strength loss. However, female REX-albuterol subjects fared best throughout the 40-day period and incurred significant unloaded knee extensor strength gains. Differences in strength changes between male and female REX-albuterol subjects was likely due to the higher relative dosage administered to the latter, as body mass showed a gender (i.e., men > women) effect. Future research may elucidate the ideal dose-response relationship for REX-albuterol treatment for use aboard manned space flights and in other disuse models. Coaches and practitioners should carefully examine their sport-governing bodies' rules on albuterol administration and give the drug only if an athlete's health warrants such treatment.

Albuterol and exercise effects on ankle extensor strength during 40 days of unloading.

INTRODUCTION: In-flight ankle extensor (AEXT) strength losses are an impediment to long-term space travel. The current study examines if albuterol helps resistance exercise (RE) abate AEXT strength loss incurred over a 40-d unloading period. METHODS: All subjects (21 men, M; 15 women, W) performed unilateral limb suspension (ULLS) and exercised on a flywheel ergometer (FERG) with their otherwise unloaded AEXT. With a double-blind randomization assignment, subjects either received placebo (PLA, lactose) or albuterol (ALB, 16 mg x d(-1)) via four daily capsule doses with no crossover. FERG calf press workouts done 3 d x wk(-1) provided concentric and eccentric total work (CTW, ETW) and average power (CAP, EAP) measures. Workout data from the 40-d period were averaged and partitioned into four consecutive 10-d periods. Data were compared with a 2 (gender) x 2 (treatment) x 4 (time) MANCOVA, with day 0 AEXT strength measurements and a drug/body mass ratio as covariates. RESULTS: CTW and ETW days 11-20, 21-30, and 31-40, as well as CAP and EAP days 11-20 and 21-30 showed the following significant results: ALB-W > ALB-M, PLA-M > PLA-W. CAP and EAP days 31-40 showed the following significant results: ALB-W, ALB-M, PLA-M > PLA-W. DISCUSSION: The combined RE-albuterol treatment most likely evoked unloaded AEXT strength gains in women due to heightened myofibril sensitivity for Ca+2. Despite a drug/body mass covariate, gender-related differences should be interpreted with caution. Future work should compare absolute and relative beta2 agonist dosages on gender-related muscle mass and strength changes.

Antisense-mediated reduction in ADC activity causes minor alterations in the alkaloid profile of cultured hairy roots and regenerated transgenic plants of Nicotiana tabacum.

In species of the genus Nicotiana, as in most plants, the important polyamine precursor putrescine can be derived from the amino acids ornithine and/or arginine via the activity of ornithine decarboxylase (ODC) and/or arginine decarboxylase (ADC), respectively. Nicotiana species also utilize putrescine to provide the pyrollidine ring of the defensive alkaloid nicotine and its derivatives. Previous biochemical studies, involving callus tissues cultured in vitro, suggested that the ADC-mediated route to putrescine is used preferentially to provide the putrescine that is utilized for nicotine synthesis in N. tabacum. To ascertain if this is the case in N. tabacum plants, where nicotine synthesis takes place exclusively in roots, we used an antisense approach to diminish ADC activity in transformed roots which were cultured in vitro. Several independent lines were recovered possessing markedly reduced levels of ADC transcript and ADC activity compared to controls. Transcript levels of other genes in this general area of metabolism, including ODC, were not altered as a result of the antisense-mediated downregulation of ADC. Concentrations of nicotine were comparable in antisense-ADC and control hairy root lines throughout most of their respective culture cycles, except at the latter stages of growth when the nicotine content of antisense-ADC hairy root lines was observed to be approximately 20% lower than in controls. Levels of anatabine, the second most abundant alkaloid typically found in N. tabacum, which is not derived from putrescine, were slightly elevated in two antisense-ADC hairy root lines at the latter stages of their culture cycles compared to controls. Comparison of alkaloid levels in leaves of transgenic plants that were regenerated from separate antisense-ADC and control transformed root lines indicated that the former possessed slightly elevated levels of anatabine but did not contain average levels of leaf nicotine that were different from that of controls. Our overall conclusion is that the ADC mediated route to putrescine plays a role, but is not of prime importance, in providing the pyrollidine ring which is used for nicotine synthesis in cultured hairy roots of N. tabacum and in roots of healthy greenhouse-grown plants.

Initiating or changing to a fixed-dose combination of Fluticasone propionate/Formoterol over Fluticasone propionate/Salmeterol: A real-life effectiveness and cost impact evaluation.

OBJECTIVE: Asthma has a substantial impact on quality of life and health care resources. The identification of a more cost-effective, yet equally efficacious, treatment could positively influence the economic burden of this disease. Fluticasone propionate/Formoterol (FP/FOR) may be as effective as Fluticasone Salmeterol (FP/SAL). We evaluated non-inferiority of asthma control in terms of the proportion of patients free from exacerbations, and conducted a cost impact analysis. METHODS: This historical, matched cohort database study evaluated two treatment groups in the Optimum Patient Care Research Database in the UK: 1) an FP/FOR cohort of patients initiating treatment with FP/FOR or changing from FP/SAL to FP/FOR and; 2) an FP/SAL cohort comprising patients initiating, or remaining on FP/SAL pMDI combination therapy. The main outcome evaluated non-inferiority of effectiveness (defined as prevention of severe exacerbations, lower limit of the 95% confidence interval (CI) of the mean difference between groups in patient proportions with no exacerbations is -3.5% or higher) in patients treated with FP/FOR versus FP/SAL. RESULTS: After matching 1:3, we studied a total of 2472 patients: 618 in the FP/FOR cohort (174 patients initiated on FP/FOR and 444 patients changed to FP/FOR) and 1854 in the FP/SAL cohort (522 patients initiated FP/SAL and 1332 continued FP/SAL). The percentage of patients prescribed FP/FOR met non-inferiority as the adjusted mean difference in proportion of no severe exacerbations (95%CI) was 0.008 (-0.032, 0.047) between the two cohorts. No other significant differences were observed except acute respiratory event rates, which were lower for patients prescribed FP/FOR (rate ratio [RR] 0.82, 95% CI 0.71, 0.94). CONCLUSIONS: Changing to, or initiating FP/FOR combination therapy, is associated with a non-inferior proportion of patients who are severe exacerbation-free at a lower average annual cost compared with continuing or initiating treatment with FP/SAL.

Albuterol aids resistance exercise in reducing unloading-induced ankle extensor strength losses.

While resistance exercise (REX) reduces ankle extensor (AE) mass and strength deficits during short-term unloading; additional treatments, concurrently administered with REX, are required to attenuate the greater losses seen with longer unloading periods. Subjects performed left leg REX, which otherwise refrained from ambulatory and weight-bearing activity for 40 days, while randomized to a capsule (placebo, albuterol) dosing regimen with no crossover to note whether albuterol helps REX mitigate unloading-induced AE losses. A third group of subjects served as unloaded controls. On days 0, 20, and 40, the following data were collected from the left leg: calf cross-sectional area and AE strength measures. Cross-sectional area was estimated using anthropometric methodology, whereas AE strength data were obtained from eight unilateral calf-press repetitions on an inertial-based REX device. Repeated-measures mixed-factorial 3 x 3 analyses of covariance, with day 0 values as a covariate, revealed group x time interactions for the strength variables eccentric total work (ETW) and average power (EAP). Tukey's honestly significant difference shows REX-placebo subjects incurred significant ETW and EAP losses by day 40, whereas the REX-albuterol treatment evoked strength gains to those same variables without concurrent muscle accretion. Corresponding concentric variables did not display similar changes. Day 40 control data significantly declined for many variables; relative to the REX-albuterol treatment, some losses were significant after 20 days. ETW and EAP gains to unloaded AE may be due to one or more mechanisms. Continued research identifying mechanisms responsible for such changes, as well as the safety of REX-albuterol administration in other models, is warranted.

Alkaloid production and capacity for methyljasmonate induction by hairy roots of two species in Tribe Anthocercideae, family Solanaceae.

In addition to producing medicinally important tropane alkaloids, some species in the mainly Australian Solanaceous tribe Anthocercideae, sister to genus Nicotiana, are known to also contain substantial levels of the pyridine alkaloids nicotine and nornicotine. Here, we demonstrate that axenic hairy root cultures of two tribe Anthocercideae species, Cyphanthera tasmanica Miers and Anthocercis ilicifolia ssp. ilicifolia Hook, contain considerable amounts of both nicotine and nornicotine (~0.5-1% DW), together with lower levels of the tropane alkaloid hyoscyamine (<0.2% DW). Treatment of growing hairy roots of both species with micromolar levels of the wound stress hormone methyl-jasmonate (MeJa) led to significant increases (P<0.05) in pyridine alkaloid concentrations but not of hyoscyamine. Consistent with previous studies involving Nicotiana species, we also observed that transcript levels of key genes required for pyridine alkaloid synthesis increased in hairy roots of both Anthocercideae species following MeJa treatment. We hypothesise that wound-associated induction of pyridine alkaloid synthesis in extant species of tribe Anthocercideae and genus Nicotiana was a feature of common ancestral stock that existed before the separation of both lineages ~15million years ago.

Albuterol helps resistance exercise attenuate unloading-induced knee extensor losses.

INTRODUCTION: While resistance exercise (REX) attenuates knee extensor (KE) mass and strength deficits during short-term unloading, additional treatments concurrently administered with REX are required to reduce the greater losses seen with longer periods of unloading. METHODS: To determine whether albuterol helps REX attenuate unloading-induced KE losses, two groups of subjects strength trained their left thigh three times per week, and otherwise refrained from ambulatory and weight-bearing activity for 40 d while receiving a capsule dosing treatment (albuterol, placebo) with no crossover. A third group served as unloaded controls (CTRL). On days 0, 20, and 40, the following data were collected from the nonweight-bearing (left) thigh: cross-sectional area (CSA); integrated electromyography (IEMG); and concentric and eccentric KE strength measures. Thigh CSA was estimated using anthropometric methodology. IEMG was used to provide root mean square (RMS) values from submaximal (100 nm) and maximal isometric contractions. Concentric and eccentric strength were measured from eight-repetition unilateral leg press sets. RESULTS: Repeated-measures mixed-factorial 3 x 3 ANCOVAs with day 0 values as a covariate showed group by time interactions for concentric and eccentric total work (CTW, ETW). Tukey's post hoc test showed REX-albuterol evoked significant (p < 0.05) day 40 CTW and ETW gains vs. within-group day 0 and within-time REX-placebo and CTRL values. By days 20 and 40, CTRL subjects incurred significant decrements. CONCLUSIONS: Albuterol augmented the effects of REX to increase CTW and ETW. Research identifying possible mechanisms responsible for such changes, as well as the safety of REX-albuterol administration in other models, is warranted.

The yield of essential oils in Melaleuca alternifolia (Myrtaceae) is regulated through transcript abundance of genes in the MEP pathway.

Medicinal tea tree (Melaleuca alternifolia) leaves contain large amounts of an essential oil, dominated by monoterpenes. Several enzymes of the chloroplastic methylerythritol phosphate (MEP) pathway are hypothesised to act as bottlenecks to the production of monoterpenes. We investigated, whether transcript abundance of genes encoding for enzymes of the MEP pathway were correlated with foliar terpenes in M. alternifolia using a population of 48 individuals that ranged in their oil concentration from 39 -122 mg x g DM(-1). Our study shows that most genes in the MEP pathway are co-regulated and that the expression of multiple genes within the MEP pathway is correlated with oil yield. Using multiple regression analysis, variation in expression of MEP pathway genes explained 87% of variation in foliar monoterpene concentrations. The data also suggest that sesquiterpenes in M. alternifolia are synthesised, at least in part, from isopentenyl pyrophosphate originating from the plastid via the MEP pathway.

RNAi-mediated down-regulation of ornithine decarboxylase (ODC) impedes wound-stress stimulation of anabasine synthesis in Nicotiana glauca.

Unlike most Nicotiana species, leaf tissues of the globally significant weed Nicotiana glauca Grah. (Argentinian tree tobacco) contains anabasine as the main component of its alkaloid pool, with concentrations typically increasing several fold in response to wounding of plants. The Δ(1)-piperidinium ring of anabasine is synthesised from cadaverine, via the decarboxylation of lysine, however the identity of the protein catalysing this reaction remains unknown. Recent studies indicate that ornithine decarboxylase (ODC), an enzyme involved in the synthesis of the diamine putrescine, may also possess LDC activity. Previously we found that ODC transcript is markedly up-regulated in leaves of N. glauca in response to wounding. In order to examine the role of ODC in the synthesis of anabasine in N. glauca, transcript levels were constitutively down-regulated in hairy root cultures and transgenic plants via the introduction of a CaMV35S driven ODC-RNAi construct. In addition to the anticipated marked reduction in nicotine concentrations, demonstrating that the ODC-RNAi construct was functioning in vivo, we observed that N. glauca ODC-RNAi hairy root cultures had a significantly diminished capacity to elevate anabasine synthesis in response to treatment with the wound-associated hormone methyl jasmonate, when compared to vector-only controls. We observed also that ODC-RNAi transgenic plants had significantly reduced ability to increase anabasine concentrations following removal of the plant apex. We conclude that ODC does have an important role in enabling N. glauca to elevate levels of anabasine in response to wound-associated stress.

Increased biomass, seed yield and stress tolerance is conferred in Arabidopsis by a novel enzyme from the resurrection grass Sporobolus stapfianus that glycosylates the strigolactone analogue GR24.

Isolation of gene transcripts from desiccated leaf tissues of the resurrection grass, Sporobolus stapfianus, resulted in the identification of a gene, SDG8i, encoding a Group 1 glycosyltransferase (UGT). Here, we examine the effects of introducing this gene, under control of the CaMV35S promoter, into the model plant Arabidopsis thaliana. Results show that Arabidopsis plants constitutively over-expressing SDG8i exhibit enhanced growth, reduced senescence, cold tolerance and a substantial improvement in protoplasmic drought tolerance. We hypothesise that expression of SDG8i in Arabidopsis negatively affects the bioactivity of metabolite/s that mediate/s environmentally-induced repression of cell division and expansion, both during normal development and in response to stress. The phenotype of transgenic plants over-expressing SDG8i suggests modulation in activities of both growth- and stress-related hormones. Plants overexpressing the UGT show evidence of elevated auxin levels, with the enzyme acting downstream of ABA to reduce drought-induced senescence. Analysis of the in vitro activity of the UGT recombinant protein product demonstrates that SDG8i can glycosylate the synthetic strigolactone analogue GR24, evoking a link with strigolactone-related processes in vivo. The large improvements observed in survival of transgenic Arabidopsis plants under cold-, salt- and drought-stress, as well as the substantial increases in growth rate and seed yield under non-stress conditions, indicates that overexpression of SDG8i in crop plants may provide a novel means of increasing plant productivity.

Effects of PEG-induced osmotic stress on growth and dhurrin levels of forage sorghum.

Sorghum (Sorghum bicolor L. Moench) is a valuable forage crop in regions with low soil moisture. Sorghum may accumulate high concentrations of the cyanogenic glucoside dhurrin when drought stressed resulting in possible cyanide (HCN) intoxication of grazing animals. In addition, high concentrations of nitrate, also potentially toxic to ruminants, may accumulate during or shortly after periods of drought. Little is known about the degree and duration of drought-stress required to induce dhurrin accumulation, or how changes in dhurrin concentration are influenced by plant size or nitrate metabolism. Given that finely regulating soil moisture under controlled conditions is notoriously difficult, we exposed sorghum plants to varying degrees of osmotic stress by growing them for different lengths of time in hydroponic solutions containing polyethylene glycol (PEG). Plants grown in medium containing 20% PEG (-0.5 MPa) for an extended period had significantly higher concentrations of dhurrin in their shoots but lower dhurrin concentrations in their roots. The total amount of dhurrin in the shoots of plants from the various treatments was not significantly different on a per mass basis, although a greater proportion of shoot N was allocated to dhurrin. Following transfer from medium containing 20% PEG to medium lacking PEG, shoot dhurrin concentrations decreased but nitrate concentrations increased to levels potentially toxic to grazing ruminants. This response is likely due to the resumption of plant growth and root activity, increasing the rate of nitrate uptake. Data presented in this article support a role for cyanogenic glucosides in mitigating oxidative stress.

The effect of plant tissue and vaccine formulation on the oral immunogenicity of a model plant-made antigen in sheep.

Antigen-specific antibody responses against a model antigen (the B subunit of the heat labile toxin of enterotoxigenic Escherichia coli, LTB) were studied in sheep following oral immunisation with plant-made and delivered vaccines. Delivery from a root-based vehicle resulted in antigen-specific immune responses in mucosal secretions of the abomasum and small intestine and mesenteric lymph nodes. Immune responses from the corresponding leaf-based vaccine were more robust and included stimulation of antigen-specific antibodies in mucosal secretions of the abomasum. These findings suggest that oral delivery of a plant bioencapsulated antigen can survive passage through the rumen to elicit mucosal and systemic immune responses in sheep. Moreover, the plant tissue used as the vaccine delivery vehicle affects the magnitude of these responses.

An interspecific Nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines.

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs.