Ruminal protein degradability of a range of tropical pastures

The rumen degradability parameters of the diet selected by two to four oesophageal-fistulated Brahman steers grazing a range of tropical pastures were determined by incubation of extrusa in nylon bags suspended in the rumen of rumen-fistulated (RF) Brahman steers. The effective protein degradability (Edg) was determined by measuring the rate of disappearance of neutral detergent insoluble nitrogen (NDIN) less acid detergent insoluble nitrogen (ADIN) in the incubated extrusa. Six to eight RF steers also grazed each of the pastures along with the oesophageal-fistulated steers, to allow determination of key rumen parameters and rumen particulate matter fractional outflow rates (FOR). The seven pastures studied included: native tropical grass (C4) pasture (major species Heteropogon contortus and Bothriochloa bladhii), studied in the early wet (NPEW), the wet/dry transition (NPT) and the dry (NPD) seasons; introduced tropical grass (C4) pasture (Bothriochloa insculpta), studied in the mid wet season (BB); the introduced tropical legumes (C3), Lablab purpureus (LL) and Clitoria ternatea (BP); and the temperate grass (C3) pasture, ryegrass (Lolium multiflorum, RG). Using the measured particle FOR values in calculations, the Edg estimates were very high for both C4 and C3 species: 0.82–0.91 and 0.95–0.98 g/g crude protein (CP), respectively. Substitution of an assumed FOR (kp = 0.02/h) for the measured values for each pasture type did not markedly affect estimates of Edg. However, C4 tropical grasses had much lower effective rumen degradable protein (ERDP) fractions (23–66 g/kg DM) than the C3 pasture species RG and LL (356 and 243 g/kg DM, respectively). This was associated with a lower potential degradability and degradation rate of organic matter (OM) in sacco, lower in vitro organic matter digestibility (IVOMD) and CP concentrations in the extrusa, and lower ammonia-N and branched-chain fatty acid concentrations in rumen fluid for the tropical grasses. As tropical grass pastures senesced, there was a decline in Edg, the ERDP and rumen undegradable protein (UDP) fractions, the potential degradability and degradation rate of OM and the IVOMD. These results provide useful data for estimating protein supply to cattle grazing tropical pastures.

Differing mechanisms of simple nitrile formation on glucosinolate degradation in Lepidium sativum and Nasturtium officinale seeds.

Glucosinolates are sulphur-containing glycosides found in brassicaceous plants that can be hydrolysed enzymatically by plant myrosinase or non-enzymatically to form primarily isothiocyanates and/or simple nitriles. From a human health perspective, isothiocyanates are quite important because they are major inducers of carcinogen-detoxifying enzymes. Two of the most potent inducers are benzyl isothiocyanate (BITC) present in garden cress (Lepidium sativum), and phenylethyl isothiocyanate (PEITC) present in watercress (Nasturtium officinale). Previous studies on these salad crops have indicated that significant amounts of simple nitriles are produced at the expense of the isothiocyanates. These studies also suggested that nitrile formation may occur by different pathways: (1) under the control of specifier protein in garden cress and (2) by an unspecified, non-enzymatic path in watercress. In an effort to understand more about the mechanisms involved in simple nitrile formation in these species, we analysed their seeds for specifier protein and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of different iron species, specific chelators and various heat treatments. We confirmed that simple nitrile formation was predominantly under specifier protein control (thiocyanate-forming protein) in garden cress seeds. Limited thermal degradation of the major glucosinolate, glucotropaeolin (benzyl glucosinolate), occurred when seed material was heated to >120 degrees C. In the watercress seeds, however, we show for the first time that gluconasturtiin (phenylethyl glucosinolate) undergoes a non-enzymatic, iron-dependent degradation to a simple nitrile. On heating the seeds to 120 degrees C or greater, thermal degradation of this heat-labile glucosinolate increased simple nitrile levels many fold.

Key Role of Fe2+ in Epithiospecifier Protein Activity.

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins such as epithiospecifier proteins (ESPs). ESPs promote the formation of epithionitriles from terminal alkenyl glucosinolates and, as recent evidence suggests, simple nitriles at the expense of isothiocyanates. From a human health perspective isothiocyanates are the most important because they are major inducers of carcinogen-detoxifying enzymes. Fe2+ is an essential factor in ESP activity, although several recent studies have highlighted discrepancies in the understanding of the ESP-iron interaction. To investigate further the role iron species play in regulating ESP activity, four ESP-containing seedpowders were analyzed for ESP and myrosinase activities, endogenous iron content, and glucosinolate degradation products after the addition of iron species, specific chelators, and reducing agents. For the first time this paper shows the effect of these additions on the hydrolysis of individual glucosinolates that constitute the total pool. Aged seeds and 3-day seedlings were also tested to investigate the effects of seed storage and early plant development on iron levels and ESP activity. The four ESP-containing plant systems tested gave two distinctive responses, thus providing strong evidence that ESPs vary markedly in their Fe2+ requirement for activity. The results also indicated that reduction of ferric to ferrous iron drives variations in ESP activity during early plant development. The reverse oxidation reaction provided a convincing explanation for the loss of ESP activity during seed storage. Aged seeds produced seedlings with substantially lower ESP activity, and there was a concomitant loss in germination rate. It was concluded that manipulation of endogenous iron levels of ESP-containing plants could increase the conversion of glucosinolates to isothiocyanates and enhance potential health benefits.

Digestion of forages in the rumen is increased by the amount but not the type of protein supplement

Three polyester bag experiments were conducted with fistulated Bos indicus steers to determine the effect of the amount and type of nitrogen (N) supplement on the digestion rate of forages different in quality. In Experiment 1, test substrates were incubated in polyester bags in the rumen of steers fed ryegrass, pangola grass, speargrass and Mitchell grass hays in a 4 by 4 Latin-square design. In Experiment 2, test substrates were incubated in polyester bags in the rumen of steers fed speargrass hay supplemented with urea and ammonium sulfate (US), branched-chain amino acids with US (USAA), casein, cottonseed meal, yeast and Chlorella algae in a 7 by 3 incomplete Latin-square design. In Experiment 3, test substrates were incubated in polyester bags in the rumen of steers fed Mitchell grass hay supplemented with increasing amounts of US or Spirulina algae (Spirulina platensis). The test substrates used in all experiments were speargrass, Mitchell grass, pangola grass or ryegrass hays. Digestion rate of the ryegrass substrate was higher than that of the speargrass substrate (P < 0.05) in Experiment 1. Supplementation with various N sources increased the degradation rate and effective degradability of all incubated substrates above that apparent in Control steers (P < 0.05; Experiment 2). Supplementation of US and Spirulina increased degradation rate and effective degradability of ryegrass, pangola grass and Mitchell grass substrates above that apparent in Control steers (P < 0.05; Experiment 3). However, there was no further response on digestion rate of the substrates in increasing supplementation levels either for US or Spirulina. In conclusion, rate of digestion was affected by forage physical and anatomical properties. Supplementation with various N sources increased rate of digestion when the Control forage ration was very low in N but once a minimum level of N supplementation was reached, irrespective of form of N or other potential growth factors, there was no further increase in rate of digestion.

Nucleotide Degradation Profiles of Iced Barramundi (Lates calcarifer) and Nile Perch (Lates niloticus) Muscle

Adenine nucleotides and their related compounds were determined in muscle extracts from two species of fish that were stored in ice after thawing. The fish were the closely related species, Australian barramundi (Lates calcarifer ) and Kenyan Nile perch (Lates niloticus ) which had different process histories. For all samples, adenine nucleotides did not exceed 6% of the total nucleotide pool. Inosine monophosphate (IMP) decreased steadily with storage. Hypoxanthine (Hx) was the major product of adenosine triphosphate (ATP) degradation in both barramundi and Nile perch, showing a steady increase with days of iced storage. The Hx level did not reach a maximum during the 9d storage period. The K-value also increased regularly with time of storage and for the later stages (i.e., 7 and 9d) and was significantly different (P < 0.01) for the two species. The iced storage life of these typical samples of barramundi and Nile perch was estimated to be 3d after thawing using a K-value of < 30% to indicate excellent quality. Despite the differences in process history the nucleotide profiles were remarkably similar during storage. This precludes the use of nucleotide levels as a means of differentiating between these species.

Vaccination with an Insulin-like Growth Factor Binding Protein-1 (IGFBP-1)-bBased Vaccine Improves Growth in Feed-Restricted Brahman Steers

Dry-season weight loss in grazing cattle in northern Australia has been attenuated using a number of strategies (Hunter and Vercoe, 1987, Sillence et al. 1993, Gazzola and Hunter, 1999). Furthermore, the potential to improve efficiency of feed utilisation (and thus, dry-season performance) in ruminants through conventional modulation of the insulin-like growth factor (IGF) axis (Oddy and Owens, 1997, Hill et al., 1999) and through immunomodulation of the IGF axis (Hill et al., 1998a,b) has been demonstrated. The present study investigated the use of a vaccine directed against IGFBP-1 in Brahman steers which underwent a period of nutritional restriction followed by a return to wet-season grazing.

Sustaining productivity of a Vertosol at Warra, Queensland, with fertilisers, no-tillage or legumes. 8. Effect of duration of lucerne ley on soil nitrogen and water, wheat yield and protein.

Soil nitrogen (N) supply in the Vertosols of southern Queensland, Australia has steadily declined as a result of long-term cereal cropping without N fertiliser application or rotations with legumes. Nitrogen-fixing legumes such as lucerne may enhance soil N supply and therefore could be used in lucerne-wheat rotations. However, lucerne leys in this subtropical environment can create a soil moisture deficit, which may persist for a number of seasons. Therefore, we evaluated the effect of varying the duration of a lucerne ley (for up to 4 years) on soil N increase, N supply to wheat, soil water changes, wheat yields and wheat protein on a fertility-depleted Vertosol in a field experiment between 1989 and 1996 at Warra (26degrees 47'S, 150degrees53'E), southern Queensland. The experiment consisted of a wheat-wheat rotation, and 8 treatments of lucerne leys starting in 1989 (phase 1) or 1990 (phase 2) for 1,2,3 or 4 years duration, followed by wheat cropping. Lucerne DM yield and N yield increased with increasing duration of lucerne leys. Soil N increased over time following 2 years of lucerne but there was no further significant increase after 3 or 4 years of lucerne ley. Soil nitrate concentrations increased significantly with all lucerne leys and moved progressively downward in the soil profile from 1992 to 1995. Soil water, especially at 0.9-1.2 m depth, remained significantly lower for the next 3 years after the termination of the 4 year lucerne ley than under continuous wheat. No significant increase in wheat yields was observed from 1992 to 1995, irrespective of the lucerne ley. However, wheat grain protein concentrations were significantly higher under lucerne-wheat than under wheat wheat rotations for 3-5 years. The lucerne yield and soil water and nitrate-N concentrations were satisfactorily simulated with the APSIM model. Although significant N accretion occurred in the soil following lucerne leys, in drier seasons, recharge of the drier soil profile following long duration lucerne occurred after 3 years. Consequently, 3- and 4-year lucerne-wheat rotations resulted in more variable wheat yields than wheat-wheat rotations in this region. The remaining challenge in using lucerne-wheat rotations is balancing the N accretion benefits with plant-available water deficits, which are most likely to occur in the highly variable rainfall conditions of this region.

Protein supplementation and growth enhancer strategies in successive years to maximise growth of Brahman cross steers grazing sown grass pastures on brigalow lands

A strategy comprising a winter/spring protein supplement, rumen modifier and hormonal growth promotant (Compudose 400) was used in either the first year (Tl), second year (T2), or in both years (T1+2) following weaning in Brahman cross steers as a means of increasing liveweight gain up to 2.5 years of age. T2 produced the heaviest final liveweight (544.7 kg) and highest overall liveweight gain (366.7 kg), but these were not significantly different from T1 (538.6 kg; 360.9 kg), or T1+2 (528.7 kg; 349.3 kg). However, final liveweight and overall liveweight gains of T1 and T2 but not T1+2 were significantly greater than for untreated (C) steers (504.9 kg; 325.2 kg, both P < 0.05). Regardless of the strategy imposed, liveweight and liveweight gain were enhanced, however final liveweights in each treatment were below the preferred minimum target liveweight (570-580 kg) for premium export markets. Treatment in both years gave no benefit over treatment in 1 year only. 19th Biennial Conference. 5-9 July 1992. LaTrobe University, Melbourne.

Peanut stripe potyvirus resistance in peanut (Arachis hypogaea L.) plants carrying viral coat protein gene sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardmentwith the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.

Temporal expression of G-protein-coupled receptor 54 (GPR54), gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus.

The G-protein-coupled receptor 54 (muGPR54) cDNA was cloned from the brain of the grey mullet, and its expression level, as well as those of the gonadotropin-releasing hormones (GnRH1, GnRH2, GnRH3) and dopamine receptor D2 (drd2), in the brain, pituitary and ovary of pubertal fish (early, intermediate, advanced) were determined by real-time quantitative RT-PCR (QPCR). The muGPR54 cDNA has an open reading frame of 1140 bp with a predicted 380 amino acid peptide, containing seven putative transmembrane domains and putative N-glycosylation and protein kinase C phosphorylation sites. QPCR results showed that the early stage of puberty in grey mullet is characterized by significantly high levels of expression of GPR54, GnRH and drd2 in the brain relative to the intermediate and advanced stages, except for GnRH1 that increased at the advanced stage of puberty. In the pituitary, drd2 expression declined significantly at the advanced stage relative to levels at the intermediate stage. Ovarian expression of GPR54 significantly increased from the intermediate stage of puberty relative to the early stage while that of GnRH1 acutely increased at the advanced stage of puberty. The ovarian expression of drd2 decreased as puberty progressed, but the changes were not significant. The results suggest the possible role of GPR54 and GnRH in positively regulating pubertal development in grey mullet and the dopaminergic inhibition of reproductive function mediated by drd2.

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

Epithiospecifier protein activity in broccoli: The link between terminal alkenyl glucosinolates and sulphoraphane nitrile

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs (non-catalytic cofactors of myrosinase) promote the formation of epithionitriles from terminal alkenyl glucosinolates and as recent evidence suggests, simple nitriles at the expense of isothiocyanates. The ratio of ESP activity to myrosinase activity is crucial in determining the proportion of these nitriles produced on hydrolysis. Sulphoraphane, a major isothiocyanate produced in broccoli seedlings, has been found to be a potent inducer of phase 2 detoxification enzymes. However, ESP may also support the formation of the non-inductive sulphoraphane nitrile. Our objective was to monitor changes in ESP activity during the development of broccoli seedlings and link these activity changes with myrosinase activity, the level of terminal alkenyl glucosinolates and sulphoraphane nitrile formed. Here, for the first time, we show ESP activity increases up to day 2 after germination before decreasing again to seed activity levels at day 5. These activity changes paralleled changes in myrosinase activity and terminal alkenyl glucosinolate content. There is a significant relationship between ESP activity and the formation of sulforaphane nitrile in broccoli seedlings. The significance of these findings for the health benefits conferred by eating broccoli seedlings is briefly discussed.

Atrazine degradation and transport in runoff on a Black Vertosol

In Australia communities are concerned about atrazine being detected in drinking water supplies. It is important to understand mechanisms by which atrazine is transported from paddocks to waterways if we are to reduce movement of agricultural chemicals from the site of application. Two paddocks cropped with grain sorghum on a Black Vertosol were monitored for atrazine, potassium chloride (KCl) extractable atrazine, desethylatrazine (DEA), and desisopropylatrazine (DIA) at 4 soil depths (0-0.05, 0.05-0.10, 0.10-0.20, and 0.20-0.30 m) and in runoff water and runoff sediment. Atrazine + DEA + DIA (total atrazine) had a half-life in soil of 16-20 days, more rapid dissipation than in many earlier reports. Atrazine extracted in dilute potassium chloride, considered available for weed control, was initially 34% of the total and had a half-life of 15-20 days until day 30, after which it dissipated rapidly with a half life of 6 days. We conclude that, in this region, atrazine may not pose a risk for groundwater contamination, as only 0.5% of applied atrazine moved deeper than 0.20 m into the soil, where it dissipated rapidly. In runoff (including suspended sediment) atrazine concentrations were greatest during the first runoff event (57 days after application) (85 μg/L) and declined with time. After 160 days, the total atrazine lost in runoff was 0.4% of the initial application. The total atrazine concentration in runoff was strongly related to the total concentration in soil, as expected. Even after 98% of the KCl-extractable atrazine had dissipated (and no longer provided weed control), runoff concentrations still exceeded the human health guideline value of 40 μg/L. For total atrazine in soil (0-0.05 m), the range for coefficient of soil sorption (Kd) was 1.9-28.4 mL/g and for soil organic carbon sorption (KOC) was 100-2184 mL/g, increasing with time of contact with the soil and rapid dissipation of the more soluble, available phase. Partition coefficients in runoff for total atrazine were initially 3, increasing to 32 and 51 with time, values for DEA being half these. To minimise atrazine losses, cultural practices that maximise rain infiltration, and thereby minimise runoff, and minimise concentrations in the soil surface should be adopted.