[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis transparent testa (tt) mutant tt19-4 shows reduced seed coat colour, but stains darkly with DMACA and accumulates anthocyanins in aerial tissues. Positional cloning showed that tt19-4 was allelic to tt19-1 and has a G-to-T mutation in a conserved 3'-domain in the TT19-4 gene. Soluble and unextractable seed proanthocyanidins and hydrolysis of unextractable proanthocyanidin differ between wild-type Col-4 and both mutants. However, seed quercetins, unextractable proanthocyanidin hydrolysis, and seedling anthocyanin content, and flavonoid gene expression differ between tt19-1 and tt19-4. Transformation of tt19-1 with a TT19-4 cDNA results in vegetative anthocyanins, whereas TT19-4 cDNA cannot complement the proanthocyanidin and pale seed coat phenotype of tt19-1. Both recombinant TT19 and TT19-4 enzymes are functional GSTs and are localized in the cytosol, but TT19 did not function with wide range of flavonoids and natural products to produce conjugation products. We suggest that the dark seed coat of Arabidopsis is related to soluble proanthocyanidin content and that quercetin holds the key to the function of TT19. In addition, TT19 appears to have a 5' GSH-binding domain influencing both anthocyanin and proanthocyanidin accumulation and a 3' domain affecting proanthocyanidin accumulation by a single amino acid substitution.
Plant Cell and Environment 11/2010; 34(3):374-88. · 5.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Structures for nine compounds were elucidated in seed coats of two genetically related Brassica carinata lines. The yellow-seeded line accumulated monomeric kaempferols, phenylpropanoids, and lignans, while extractable and unextractable proanthocyanidins and a high-performance liquid chromatography peak containing polymeric-like quercetin/lignan structures were strongly reduced. The brown-seeded line accumulated large amounts of both types of proanthocyanidins (extractable and unextractable), as well as phenylpropanoids and lignans equivalent to the amounts in the yellow-seeded seed coats, but the brown-seeded seed coats lacked kaempferols. A Brassica napus 15K oligoarray experiment indicated that yellow-seeded siliques had more extreme gene expression changes and a 2.4-fold higher number of upregulated genes than brown-seeded siliques, including a host of transcription factors and genes with unknown function. Transcripts for six flavonoid genes (CHS, F3H, FOMT, DFR, GST, and TTG1) were lower and two (F3'H and FLS) were higher in yellow-seeded siliques, but expression of CHI, PAP1, and phenylpropanoid genes was unchanged.
Journal of Agricultural and Food Chemistry 10/2010; · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Deposition and persistence of carbofuran in vegetation and grasshoppers of two prairie pastures were measured after single aerial applications at 134 g a.i per hectare in 1987 and 1988. Mean depositions on horizontal filter paper samplers were 43 1 and 64 9% of the nominal rate in the two applications Variability of deposits, measured as C V.s, were 76 4% and 86 9% Carbofuran residues in vegetation and grasshoppers were less variable than those in deposit samplers, with C V s of 40 6 and 70 6% in vegetation and 58 6 and 32 5% in grasshoppers for 1987 and 1988, respectively. Residues declined by 50% in 24 h both years from initial concentrations (3 h postspray) in vegetation of 13 3 and 10 8 μg/g Initial residues in grasshoppers averaged 2 2 μg/g in 1987 and 3.9 μg/g in 1988; the mean for both years was 2.5 μg/g Grasshoppers contained the same concentration of carbofuran 3 h after spraying, whether collected live or dead Maximum residues of 5 7 μg/g were found in grasshoppers from two of the 40 sites sampled in both years A model fitted to the data indicated that there may have been about a 24 to 44% loss of residues from vegetation, probably due to volatilization and other processes, before samples were collected at 3 h There was a decline in residues per grasshopper of 30% in 24 h both years, which was not evident when expressed as micrograms per gram because of desiccation of the dead insects The concentrations of carbofuran in the vegetation and grasshoppers were typical of aerial spraying, hence, they may be used as baseline values for estimating exposure of herbivores and msectivores to insecticide deposits from similar spray events by adjusting for rate of application Deposits measured on filter papers were positively related to concentrations in vegetation and grasshoppers Calculations of the potential effects on wildlife of ingesting sprayed grasshoppers, combined with other routes of exposure, indicated a relatively low hazard for herbivorous rodents but a relatively high hazard for insectivorous adult passerines and their young Monitoring passerines in a related field study did, however, not reveal any effects on survival within 3 d of spraying carbofuran.
[Show abstract][Hide abstract] ABSTRACT: Mallard (Anas platyrhynchos) ducklings were led for distances of 50, 150 and 300 m through plots of upland vegetation that had been sprayed with a flowable formulation of carbofuran at rates of 0, 132 and 264 g active ingredient/ha. Duckling behavior was observed and quantified immediately after exposure by using an approach-response apparatus. Brain and plasma cholinesterase (ChE) activities were determined for a subsample of ducklings from each treatment group; the remaining ducklings were monitored for growth effects. No mortality occurred during field exposure, but toxic signs were observed in ducklings moving < 150 m at both spray rates. At > 150 m, 17 and 100% of the ducklings had difficulty keeping pace due to anti-ChE toxicosis in the low and high spray plots, respectively. Duckling approach-response behavior became significantly slower with increasing spray rate and exposure distance (p = 0.0001). Inhibition of brain ChE activity was directly related to both spray rate and exposure distance; ducklings receiving maximum carbofuran exposure had 29% of the mean ChE activities of controls. Plasma ChE activity was inversely related to spray rate but was not affected by exposure distance and did not correlate well with brain ChE activity (R2 = 0.36). Subsequent growth rates were not affected in a dose-related manner.
[Show abstract][Hide abstract] ABSTRACT: The potential hazard of carbofuran-sprayed insects to clay-colored sparrows (Spizella pallida) was determined by measuring consumption of grasshoppers (Melanoplus sanguinipes) by nestlings and adults, attractiveness of dead vs. live grasshoppers, and toxicity of sprayed grasshoppers to adults. Hand-reared nestlings 8 to 10 and 14 to 22 d old ate mean quantities of grasshoppers per bird of 0.61 and 0.83 g, respectively, per bout of feeding when fed to satiety every hour from dawn to dusk. These rates of intake in the field could result in doses of 0.2 mg/kg body mass (14% of the LD50 for adults) in brief bouts of feeding if they were fed sprayed insects containing carbofuran at 2.5 μg/g, the mean concentration found after aerial application of 134 g a.i. per hectare. Nestlings 2 to 3 d old ingesting about 0.2 g grasshoppers in brief periods might receive lethal doses from carbofuran residues of 2.5 μg/g or the maximum concentration of 5.7 μg/g if they are more sensitive than adults. There was nonetheless no evidence of reduced survival among nestling passerines for 3 d following aerial spraying of carbofuran at 134 g/ha in a pasture. Captive adults preferred dead grasshoppers in trials in which live and dead grasshoppers were offered simultaneously. When six adults were provided with 24-h supplies of sprayed grasshoppers containing carbofuran at 2.6 μg/g, they showed no taste aversion and consumed quantities 24% greater than those eaten by control birds. No mortality resulted from eating the contaminated grasshoppers. Mean hopping activity by treated birds increased by 80%, relative to controls, 5 h after receiving poisoned grasshoppers, but the difference was not significant. Although captive adults consumed close to their body mass in grasshoppers daily, an LD50 of carbofuran from grasshoppers containing 2.5 μg/g would require rapid ingestion of 6.7 g, a quantity about 7x the birds' capacity for short-term ingestion. Our results, combined with observations of a related field study, suggest that adult and nestling passerines are able to tolerate the dietary exposure to carbofuran resulting from ingestion of grasshoppers sprayed at the rate of 134 g/ha. Conclusions about safety should not be drawn, however, until more field applications have been monitored.
[Show abstract][Hide abstract] ABSTRACT: The oxygen radical absorbance capacity (ORAC), antiproliferative activities and mycosporine-like amino acid (MAA) profiles of methanol extracts from two grades of dulse harvested from locations varying in UV-exposure (west vs east coasts of Grand Manan Island, NB) were determined in the present study. MAAs confirmed by LC/MS in both grades 1 (low-UV) and 2 (high-UV) dulse were palythine, shinorine, asterina-330, palythinol and porphyra-334; usujirene was present only in grade 2 dulse. ORAC values of grade 1 and 2 dulse extracts were 36.42 and 38.78 μmol Trolox/g extract. B16-F1 murine skin melanoma cell proliferation was inhibited (p < .05) by 68.5% and 91.9% by grade 1 and 2 dulse extracts at 6.0 mg/mL. The antiproliferative efficacy of grade 2 dulse was greater (p < .05) than grade 1 from 0.375 to 6.0 mg/mL. MAA differences between the grade 1 and 2 dulse extracts likely influenced the antiproliferative efficacies, despite the similar ORAC values.
[Show abstract][Hide abstract] ABSTRACT: Exposure of B. carinata seedlings to increasing concentrations of a non-physiological ion, lithium, showed significant effects on the germination rate, root length, chlorophyll content and fresh weight in brown-seeded and yellow-seeded near-isogenic lines. Metal content analysis and phytochemical profiling indicated that lithium hyper-accumulated and the lipid and phenolic composition dramatically changed in brown-seeded seedlings. Here, sinapic acid esters and chloroplast lipid were replaced by benzoate derivatives, resveratrol and oxylipins after lithium exposure. In contrast, the yellow-seeded plants maintained the same phenolic and lipid composition before and after exposure to lithium and did not tolerate the high metal concentrations tolerated by the brown-seeded line. Microarray analysis using a Brassica napus 15,000 expressed sequence tag (EST) array indicated a total of 89 genes in the brown-seeded line and 95 genes in the yellow-seeded line were differentially expressed more than 20-fold after treatment of B. carinata seedlings with lithium chloride and more than 1083 genes with expression changes greater than 2-fold. The putative functions of the differentially expressed genes included proteins involved in defense, primary metabolism, transcription, transportation, secondary metabolism, cytochrome P450, as well as proteins with unknown functions. Transcriptome changes between yellow-seeded and brown-seeded B. carinata seedlings after lithium chloride exposure indicated that the two lines responded differently to lithium treatment. The expression patterns generally supported the phytochemical data. From the results of this study, B. carinata brown-seeded germplasm showed an ability to survive under moderately high concentrations of lithium chloride (>150 mM). The ability to accumulate this metal ion suggests brown-seeded B. carinata has some potential in phytoremediation of lithium-contaminated water and soil.
[Show abstract][Hide abstract] ABSTRACT: Improving our knowledge of plant metal metabolism is facilitated by the use of analytical techniques to map the distribution of elements in tissues. One such technique is X-ray fluorescence (XRF), which has been used previously to map metal distribution in both two and three dimensions. One of the difficulties of mapping metal distribution in two dimensions is that it can be difficult to normalize for tissue thickness. When mapping metal distribution in three dimensions, the time required to collect the data can become a major constraint. In this article a compromise is suggested between two- and three-dimensional mapping using multi-angle XRF imaging.
A synchrotron-based XRF microprobe was used to map the distribution of K, Ca, Mn, Fe, Ni, Cu and Zn in whole Arabidopsis thaliana seeds. Relative concentrations of each element were determined by measuring fluorescence emitted from a 10 microm excitation beam at 13 keV. XRF spectra were collected from an array of points with 25 or 30 microm steps. Maps were recorded at 0 and 90 degrees , or at 0, 60 and 120 degrees for each seed. Using these data, circular or ellipsoidal cross-sections were modelled, and from these an apparent pathlength for the excitation beam was calculated to normalize the data. Elemental distribution was mapped in seeds from ecotype Columbia-4 plants, as well as the metal accumulation mutants manganese accumulator 1 (man1) and nicotianamine synthetase (nasx).
Multi-angle XRF imaging will be useful for mapping elemental distribution in plant tissues. It offers a compromise between two- and three-dimensional XRF mapping, as far as collection times, image resolution and ease of visualization. It is also complementary to other metal-mapping techniques. Mn, Fe and Cu had tissue-specific accumulation patterns. Metal accumulation patterns were different between seeds of the Col-4, man1 and nasx genotypes.
Annals of Botany 01/2008; 100(6):1357-65. · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Secoisolariciresinol (SECO ) is the major lignan found in flaxseed (Linum usitatissimum L.) and is present in a polymer that contains secoisolariciresinol diglucoside (SDG ). SECO, SDG and the polymer are known to have a number of health benefits, including reduction of serum cholesterol levels, delay in the onset of type II diabetes and decreased formation of breast, prostate and colon cancers. The health benefits of SECO and SDG may be partially attributed to their antioxidant properties. To better understand their antioxidant properties, SECO and SDG were oxidized using 2,2'-azobis(2-amidinopropane), an in vitro model of radical scavenging. The major lignan radical-scavenging oxidation products and their formation over time were determined. SDG was converted to four major products, which were the result of a phenoxyl radical intermediate. One of these products, a dimer of SDG, decomposed under the reaction conditions to form two of the other major products, and . SECO was converted to five major products, two of which were also the result of a phenoxyl radical intermediate. The remaining products were the result of an unexpected alkoxyl radical intermediate. The phenol oxidation products were stable under the reaction conditions, whereas two of the alcohol oxidation products decomposed. In general, only one phenol group on the lignans was oxidized, suggesting that the number of phenols per molecule may not predict radical scavenging antioxidant ability of lignans. Finally, SECO is a superior antioxidant to SDG, and it may be that the additional alcohol oxidation pathway contributes to its greater antioxidant ability.
[Show abstract][Hide abstract] ABSTRACT: The identification of genes involved in metal metabolism in plants requires the 'screening' of thousands of genetic variants. While inductively coupled plasma mass-spectroscopy has been used to identify variants with an altered total metal concentration, a more convenient high-throughput technique capable of examining individual seeds (or other tissues) would be useful. Here, the high brightness of synchrotron radiation has been utilised to examine relative metal concentrations in seeds of the genetically well characterised plant Arabidopsis thaliana. The relative concentrations of Mn, Fe, Ni, Cu and Zn in individual seeds were determined using a 500 microm x 500 microm beam. Metal concentrations were normally distributed, except where metal-containing dust contaminated the samples. Neither seed orientation nor genetic background (from three 'wild type' variants with different genetic lineages) had a significant affect on the Zn-normalised metal concentration. No advantages, such as the observation of tissue-specific metal accumulation, were obtained by using a 50 microm x 50 microm beam. A high-throughput proof-of-concept experiment was demonstrated that could be used to screen libraries of genetic variants for individuals with altered metal concentrations. Further work is required to standardise the technique before screening of libraries is possible.
[Show abstract][Hide abstract] ABSTRACT: The residues from metered doses of cypermethrin were recovered from excised portions of grasshopper cuticle to determine losses by volatilisation. Residues from similar doses were also recovered from the cuticles of intact grasshoppers and from whole body homogenates after rinsing the cuticle, to determine rates of absorption and losses from metabolism. Residues were recovered from males and females one week and three weeks after fledging in grasshoppers infected with Malameba locustae and in uninfected ones. They were maintained at 15 or 30°C and sampled from each combination of factors at 4, 8, 16, 24, 30 and 48 h after treatment. There was no measurable loss of cypermethrin by volatilisation up to 72 h after application to excised portions of cuticle at either 15 or 30°C. In all combinations of factors, more cypermethrin was recovered externally (P <0.05) from grasshoppers held at 15°C than those held at 30°C and more from infected grasshoppers than from those that were uninfected. Significantly more cypermethrin was also recovered externally from males than from females (P <0.05) and more from infected one-week-old grasshoppers than those that were three weeks old. At 15°C there was a gradual trend towards accumulation of cypermethrin internally, but at 30°C an initial trend towards accumulation was followed by one of decline. Residual cypermethrin recovered could in general be related to the negative temperature coefficient of toxicity, to a decreased sensitivity to cypermethrin in infected grasshoppers and to an increased sensitivity in older grasshoppers.
[Show abstract][Hide abstract] ABSTRACT: A new method has been developed for the extraction of volatiles from plant materials and tested on seedling tissue and mature leaves of Arabidopsis thaliana, pine needles and commercial mixtures of plant volatiles. Volatiles were extracted with n-pentane and then subjected to quick distillation at a moderate temperature. Under these conditions, compounds such as pigments, waxes and non-volatile compounds remained undistilled, while short-chain volatile compounds were distilled into a receiving flask using a high-efficiency condenser. Removal of the n-pentane and concentration of the volatiles in the receiving flask was carried out using a Vigreux column condenser prior to GC-MS. The method is ideal for the rapid extraction of low-boiling-point volatiles from small amounts of plant material, such as is required when conducting metabolic profiling or defining biological properties of volatile components from large numbers of mutant lines.
[Show abstract][Hide abstract] ABSTRACT: Dietary modification contributes significantly in reducing cardiovascular disease (CVD) risk factors including lowering cholesterol and atherosclerosis. The purpose of this study was to investigate the effects of flaxseed, a rich source of lignans, alpha-linolenic acid and soluble fiber mucilage, on the prevention of ovariectomy-induced rise in total cholesterol and atherosclerotic lesions.
Seventy-two 6-month-old female Golden Syrian hamsters were either ovariectomized (ovx) or sham-operated (sham) and randomly assigned to six groups (n = 12): sham, ovx, or ovx plus either 17beta-estradiol (E(2), 10 microg/kg body weight) or semi-purified diet adjusted for macronutrients and fiber to contain one of the three doses of flaxseed (7.5, 15, or 22.5%) for 120 days.
Ovariectomy significantly elevated plasma total-, HDL-, and free-cholesterol concentrations. Similar to estrogen, all doses of flaxseed were effective in preventing the ovx-induced rise in plasma total cholesterol. Triglyceride concentrations were significantly higher in the flax-fed hamsters. There were no significant differences in plasma non-HDL- and esterified-cholesterol among the treatment groups. Ovariectomy also increased the number of hamsters with lesions and the aortic fatty streak area. All three doses of flaxseed reduced the fatty streak area and the incidence of lesions to levels similar to the sham group.
The findings of this study show that flaxseed is beneficial in reducing plasma cholesterol and plaque formation induced by ovarian hormone deficiency.
[Show abstract][Hide abstract] ABSTRACT: Phytoestrogens offer a possible alternative to hormone replacement therapy. Flax seed contains large quantities of a phytoestrogen precursor, secoisolariciresinol diglucoside (SDG), as well as large quantities of alpha-linolenic acid; these factors may be protective against vascular disease. We have previously shown that the rise in blood pressure during mental stress is a strong predictor of atherosclerosis progression.
35 postmenopausal women with vascular disease, 62 +/- 8 years of age, were treated in a random-sequence double-blind Latin square crossover study comparing three strains of flax seed: Flanders (low in lignan and high in alpha-linolenic acid), Linola 989 (high in lignan and low in alpha-linolenic acid) and AC Linora (intermediate in both lignan and alpha-linolenic acid).
Compared to the pre-treatment baseline diet, all three strains of flax significantly reduced blood pressure during mental stress induced by a frustrating cognitive task (Stroop color-word interference task) (p = 0.004). Linola 989, the strain highest in lignan and lowest in alpha-linolenic acid, was associated with the least increase in peripheral resistance during stress, the greatest reduction in plasma cortisol during stress and the smallest increase in plasma fibrinogen during mental stress.
Flax phytoestrogens ameliorate certain responses to stress and thus may afford protection against atherosclerosis; this hypothesis should be tested in clinical trials.
Journal of the American College of Nutrition 01/2004; 22(6):494-501. · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The flavonoid profiles of 22 accessions of Camelina sativa and five other crucifer species, Crambe abyssinica, Crambe hispanica, Thlaspi arvense, Brassica napus, and Sinapis alba, were studied by a combination of liquid, paper and thin layer chromatography. Flavonoids were confirmed by comparison of their characteristics, including colour under UV light, changes to colour under UV with fuming in NH3 vapour, UV spectra and comparison of RF-values, with those of authentic standards. HPLC-mass spectroscopic data were obtained to confirm identities of several compounds. Flavonoids present in several other crucifer species were identified by TLC only. We report the accumulation of derivatives of the flavonols, quercetin, in C. sativa; quercetin and kaempferol in C. hispanica var. glabrata; quercetin, kaempferol and isorhamnetin in B. napus; and kaempferol and isorhamnetin in S. alba. Derivatives of the flavones, apigenin and luteolin, accumulate in C. abyssinica, C. hispanica var. hispanica and T. arvense leaves.
Biochemical Systematics and Ecology 11/2003; 31(11):1309-1322. · 1.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lignans have been part of both diet and herbal medicines for centuries. It is only in the last half century that phytochemists
have described the structures of the lignans. Pharmacologists have only become interested in the biological activity of lignans
in the last few decades. Much of the early interest focused on podophyllotoxin type lignans and their derivatives. Recent
literature has recorded very many new lignans or lignan derivatives with a diverse range of biological activities. In 1955,
the isolation from flax seed and the structure of the lignan derivative secoisolariciresinol diglucoside (SDG) was reported.
Possible biological activity of SDG, and the mammalian lignan metabolites, enterolactone and enterodiol, was initially reported
about 20 years later. During the next 30 years, there has been extensive research on the biological effects of both flax seed
and rye lignans since both are metabolized into the mammalian lignans. Research on the activity of lignans on breast, colon,
prostate and thyroid cancer has generally shown beneficial effects although there are some studies with either no conclusive
or negative effect. Lignans have been shown to have positive effects in lowering relative risk factors for heart disease.
Use of flax seed or SDG has been shown to have positive effects in both lupus and polycystic kidney disease models. Studies
of both type I and II diabetes models have reported positive results when using SDG. Flax seed has also been reported to be
hepatoprotective. Reproductive effects have been observed with flax seed or SDG and have been found to be dose and time related.
There are many possible mechanistic explanations for the observed bioactivities including involvement in hormonal metabolism
or availability, angiogenesis, anti-oxidation and gene suppression. Abbreviations: ALA – alpha linolenic acid; ApcMin – adenomatous polyposis coli multiple intestinal neoplasia; BBdp – BioBreeding diabetic
prone; CCl4– carbon tetrachloride; CDC – Crop Development Centre; CHD – cardiovascular heart disease; DMBA – dimethylbenzanthracene;
DNA – deoxyribonucleic acid; ER – estrogen receptor; λGT –γ-glutamyltranspeptidase; HDL – high-density lipoprotein; HMGA –
hydroxymethyl glutaric acid; IDDM – insulin dependent diabetes mellitus; LDL – low-density lipoprotein; MRL/lpr, Murine Lupus/lymphoproliferative;
MDA – malondiadehyde; NIDDM – non-insulin dependent diabetes mellitus; ORF – oxygen free radical; PAF – platelet activating
factor; PKD – Polycystic kidney disease; PEPCK – phosphoenolpyruvate carboxykinase; PMNL – polymorphonuclear monocytes; STZ
– streptozoticin; TC – total cholesterol; TG – triglycerides; SDG – secoisolariciresinol diglucoside; ZDF – Zucker diabetic
[Show abstract][Hide abstract] ABSTRACT: Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5'-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5'-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (+)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass.