Proteome-Wide Response of Dormant Caryopses of the Weed, Avena fatua, After Colonization by a Seed-Decay Isolate of Fusarium avenaceum.

Promoting seed decay is an ecological approach to reducing weed persistence in the soil seedbank. Previous work demonstrated that Fusarium avenaceum F.a.1 decays dormant Avena fatua (wild oat) caryopses and induces several defense enzyme activities in vitro. The objectives of this study were to obtain a global perspective of proteins expressed after F.a.1-caryopsis colonization by conducting proteomic evaluations on (i) leachates, soluble extrinsic (seed-surface) proteins released upon washing caryopses in buffer and (ii) proteins extracted from whole caryopses; interactions with aluminum (Al) were also evaluated in the latter study because soil acidification and associated metal toxicity are growing problems. Of the 119 leachate proteins classified as defense/stress, 80 were induced or repressed. Defense/stress proteins were far more abundant in A. fatua (35%) than in F.a.1 (12%). Avena defense/stress proteins were also the most highly regulated category, with 30% induced and 35% repressed by F.a.1. Antifungal proteins represented 36% of Avena defense proteins and were the most highly regulated, with 36% induced and 37% repressed by F.a.1. These results implicate selective regulation of Avena defense proteins by F.a.1. Fusarium proteins were also highly abundant in the leachates, with 10% related to pathogenicity, 45% of which were associated with host cell wall degradation. In whole caryopsis extracts, fungal colonization generally resulted in induction of a similar set of Avena proteins in the presence and absence of Al. Results advance the hypothesis that seed decay pathogens elicit intricate and dynamic biochemical responses in dormant seeds.

Chronic Sublethal Aluminum Exposure and Avena fatua Caryopsis Colonization Influence Gene Expression of Fusarium avenaceum F.a.1.

Fusarium avenaceum F.a.1 is a novel strain of a fungal plant pathogen capable of preferentially decaying wild oat (Avena fatua) caryopses compared with those of wheat (Triticum aestivum). Understanding the molecular mechanisms governing weed seed-pathogen interactions is crucial to developing novel weed seed suppression technologies. Additionally, wild oat often competes with wheat in regions undergoing soil acidification, which leads to increases in soluble concentrations of many metals, including aluminum (Al). There is a dearth of information regarding the gene expression responses of Fusarium species to Al toxicity, or how metal toxicity might influence caryopsis colonization. To address this, a transcriptomic approach was used to investigate molecular responses of F.a.1 during wild oat caryopsis colonization in the presence and absence of chronic, sublethal concentrations of Al (400 µM). Caryopsis colonization was associated with induction of genes related to virulence, development, iron metabolism, oxidoreduction, stress, and detoxification, along with repression of genes associated with development, transport, cell-wall turnover, and virulence. Caryopsis colonization during Al exposure resulted in the induction of genes associated with virulence, detoxification, stress, iron metabolism, oxidoreduction, and cell wall turnover, along with repression of genes associated with cell wall metabolism, virulence, development, detoxification, stress, and transcriptional regulation. Aluminum exposure in the absence of caryopses was associated with induction of genes involved in siderophore biosynthesis, secretion, uptake, and utilization, along with several other iron metabolism-related and organic acid metabolism-related genes. The siderophore-related responses associated with Al toxicity occurred concurrently with differential regulation of genes indicating disruption of iron homeostasis. These findings suggest Al toxicity is attenuated by siderophore metabolism in F.a.1. In summary, both caryopsis colonization and Al toxicity uniquely influence transcriptomic responses of F.a.1.

Physical Mapping of Peroxidase Genes and Development of Functional Markers for TaPod-D1 on Bread Wheat Chromosome 7D.

Peroxidase (POD) activity in wheat (Triticum aestivum L.) grain influences natural carotenoid pigment content and is associated with the color of flour, and processing and product quality. Here, we report the molecular characterization and physical mapping of POD genes in bread wheat. The complete genomic DNA (gDNA) sequence of two POD genes (TaPod-A2 and TaPod-D1), and the partial gDNA sequence of two additional POD genes (TaPod-A3 and TaPod-B1) from wheat were characterized using in silico cloning and validated through laboratory experiments. Using a set of 21 nullisomic-tetrasomic (NT) lines, six group-7 ditelosomic (Dt) lines, and 38 group-7 deletion (Del) lines of Chinese Spring (CS), TaPod-A2 and TaPod-D1 were found to be physically located on 0.73-0.83 and on the most distal 0.39 fraction arm length (FL) of 7AS and 7DS in cv. CS, respectively; whereas, TaPod-A3 and TaPod-B1 were assigned to the 0.40-0.49 and 0.40-0.48 FL of 7AL and 7BL, respectively. Based on single nucleotide polymorphisms (SNPs) of two alleles at the TaPod-D1 locus, two functional markers POD-7D1 and POD-7D6 were developed, amplifying 540- and 640-bp, fragments in varieties with higher and lower POD activities, respectively. A total of 224 wheat varieties were analyzed and showed a significant association between the polymorphic fragments and POD activity using POD-7D1 and POD-7D6 markers. The analysis of variance (ANOVA) indicated the average POD activities of 115 varieties with TaPod-D1a were significantly lower than 109 varieties with TaPod-D1b (P < 0.01). This study provides useful information of the POD genes in bread wheat, insight into wheat genome synteny and structure, gene-specific markers, and contributes a valuable resource for quality improvement in wheat breeding programs.

Defense Enzyme Responses in Dormant Wild Oat and Wheat Caryopses Challenged with a Seed Decay Pathogen.

Seeds have well-established passive physical and chemical defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. However, there are few studies evaluating potential biochemical defenses of dormant seeds against pathogens. Caryopsis decay by the pathogenic Fusarium avenaceum strain F.a.1 was relatively rapid in wild oat (Avena fatua L.) isoline "M73," with >50% decay after 8 days with almost no decay in wheat (Triticum aestivum L.) var. RL4137. Thus, this fungal strain has potential for selective decay of wild oat relative to wheat. To study defense enzyme activities, wild oat and wheat caryopses were incubated with F.a.1 for 2-3 days. Whole caryopses were incubated in assay reagents to measure extrinsic defense enzyme activities. Polyphenol oxidase, exochitinase, and peroxidase were induced in whole caryopses, but oxalate oxidase was reduced, in response to F.a.1 in both species. To evaluate whether defense enzyme activities were released from the caryopsis surface, caryopses were washed with buffer and enzyme activity was measured in the leachate. Significant activities of polyphenol oxidase, exochitinase, and peroxidase, but not oxalate oxidase, were leached from caryopses. Defense enzyme responses were qualitatively similar in the wild oat and wheat genotypes evaluated. Although the absolute enzyme activities were generally greater in whole caryopses than in leachates, the relative degree of induction of polyphenol oxidase, exochitinase, and peroxidase by F.a.1 was greater in caryopsis leachates, indicating that a disproportionate quantity of the induced activity was released into the environment from the caryopsis surface, consistent with their assumed role in defense. It is unlikely that the specific defense enzymes studied here play a key role in the differential susceptibility to decay by F.a.1 in these two genotypes since defense enzyme activities were greater in the more susceptible wild oat, compared to wheat. Results are consistent with the hypotheses that (1) dormant seeds are capable of mounting complex responses to pathogens, (2) a diversity of defense enzymes are involved in responses in multiple plant species, and (3) it is possible to identify fungi capable of selective decay of weed seeds without damaging crop seeds, a concept that may be applicable to weed management in the field. While earlier work on seed defenses demonstrated the presence of passive defenses, this work shows that dormant seeds are also quite responsive and capable of activating and releasing defense enzymes in response to a pathogen.

Repeatability of Mice Consumption Discrimination of Wheat (Triticum aestivum L.) Varieties across Field Experiments and Mouse Cohorts.

UNLABELLED: Whole grain wheat (Triticum aestivum L.) foods can provide critical nutrients for health and nutrition in the human diet. Potential flavor differences among varieties can be examined using consumption discrimination of the house mouse (Mus musculus L.) as a model system. This study examines consistency and repeatability of the mouse model and potentially, wheat grain flavor. A single elimination tournament design was used to measure relative consumption preference for hard red spring and hard white spring varieties across all 3 experiments in combination with 2 mouse cohorts. Fifteen replicate mice were used in 24-h trials to examine differences in preference among paired wheat varieties until an overall "winner" was established as the most highly preferred variety of wheat. In all 3 experiment-cohort combinations, the same varieties were preferred as the "winner" of both the hard red spring and hard white spring wheat varieties, Hollis and BR 7030, respectively. Despite the consistent preference for these varieties across experiments, the degree (magnitude) to which the mice preferred these varieties varied across experiments. For the hard white spring wheat varieties, the small number of varieties and confounding effects of experiment and cohort limited our ability to accurately gauge repeatability. Conversely, for the hard red spring wheat varieties, consumption preferences were consistent across experiments and mice cohorts. The single-elimination tournament model was effective in providing repeatable results in an effort to more fully understand the mouse model system and possible flavor differences among wheat varieties. PRACTICAL APPLICATION: The mouse model system used here is effective in identifying wheat varieties that may be more or less desirable to humans in whole wheat foods. The system identifies consistent differences across different mouse cohorts and crop years.

Polyphenol oxidase as a biochemical seed defense mechanism.

Seed dormancy and resistance to decay are fundamental survival strategies, which allow a population of seeds to germinate over long periods of time. Seeds have physical, chemical, and biological defense mechanisms that protect their food reserves from decay-inducing organisms and herbivores. Here, we hypothesize that seeds also possess enzyme-based biochemical defenses, based on induction of the plant defense enzyme, polyphenol oxidase (PPO), when wild oat (Avena fatua L.) caryopses and seeds were challenged with seed-decaying Fusarium fungi. These studies suggest that dormant seeds are capable of mounting a defense response to pathogens. The pathogen-induced PPO activity from wild oat was attributed to a soluble isoform of the enzyme that appeared to result, at least in part, from proteolytic activation of a latent PPO isoform. PPO activity was also induced in wild oat hulls (lemma and palea), non-living tissues that cover and protect the caryopsis. These results are consistent with the hypothesis that seeds possess inducible enzyme-based biochemical defenses arrayed on the exterior of seeds and these defenses represent a fundamental mechanism of seed survival and longevity in the soil. Enzyme-based biochemical defenses may have broader implications since they may apply to other defense enzymes as well as to a diversity of plant species and ecosystems.

The house mouse (Mus musculus L.) exerts strong differential grain consumption preferences among hard red and white spring wheat (Triticum aestivum L.) varieties in a single-elimination tournament design.

Wheat (Triticum aestivum L.) plays a central role in the health and nutrition of humans. Yet, little is known about possible flavor differences among different varieties. We have developed a model system using the house mouse (Mus musculus L.) to determine feeding preferences as a prelude to extending results to human sensory analysis. Here, we examine the application of a single-elimination tournament design to the analysis of consumption preferences of a set of hard red and hard white spring wheat varieties. A single-elimination tournament design in this case pairs 2 wheat varieties and only 1 of the 2 is advanced to further tests. Preferred varieties were advanced until an overall "winner" was identified; conversely, less desirable varieties were advanced such that an overall "loser" was identified. Hollis and IDO702 were the winner and loser, respectively, for the hard red varieties, and Clear White 515 and WA8123 were the winner and loser, respectively, for the hard white varieties. When using the more powerful protocol of 14 mice and a 4-d trial, differences in mean daily consumption preferences of 2 varieties were separated at P-values as small as 2 × 10(-8) . The single-elimination tournament design is an efficient means of identifying the most and least desirable varieties among a larger set of samples. One application for identifying the 2 extremes in preference within a group of varieties would be to use them as parents of a population to identify quantitative trait loci for preference.

Effect of processing on phenolic composition of dough and bread fractions made from refined and whole wheat flour of three wheat varieties.

This study investigated the effect of breadmaking on the assay of phenolic acids from flour, dough, and bread fractions of three whole and refined wheat varieties. Comparison of the efficacy of two commonly used methods for hydrolysis and extraction of phenoilc acids showed that yields of total phenolic acids (TPA) were 5-17% higher among all varieties and flour types when samples were directly hydrolyzed in the presence of ascorbate and EDTA as compared to the method separating free, soluble conjugates and bound, insoluble phenolic acids. Ferulic acid (FA) was the predominant phenolic acid, accounting for means of 59 and 81% of TPA among all refined and whole wheat fractions, respectively. All phenolic acids measured were more abundant in whole wheat than in refined samples. Results indicated that the total quantified phenolic acids did not change significantly when breads were prepared from refined and whole wheat flour. Thus, the potential phytochemical health benefits of total phenolic acids appear to be preserved during bread baking.

Sphingosine-1-phosphate induces pro-remodelling response in airway smooth muscle cells.

BACKGROUND: Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. OBJECTIVE: To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. METHODS: Airway smooth muscle cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and Western blotting. Receptor signalling and function were determined by mRNA knockdown and intracellular calcium mobilization experiments. RESULTS: S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P2 and S1P3 receptors activated intracellular calcium mobilization and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. CONCLUSION: S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma.

Did the house mouse (Mus musculus L.) shape the evolutionary trajectory of wheat (Triticum aestivum L.)?

Wheat (Triticum aestivum L.) is one of the most successful domesticated plant species in the world. The majority of wheat carries mutations in the Puroindoline genes that result in a hard kernel phenotype. An evolutionary explanation, or selective advantage, for the spread and persistence of these hard kernel mutations has yet to be established. Here, we demonstrate that the house mouse (Mus musculus L.) exerts a pronounced feeding preference for soft over hard kernels. When allele frequencies ranged from 0.5 to 0.009, mouse predation increased the hard allele frequency as much as 10-fold. Studies involving a single hard kernel mixed with ∼1000 soft kernels failed to recover the mutant kernel. Nevertheless, the study clearly demonstrates that the house mouse could have played a role in the evolution of wheat, and therefore the cultural trajectory of humankind.

Optimizing experimental design using the house mouse (Mus musculus L.) as a model for determining grain feeding preferences.

There is little research evaluating flavor preferences among wheat varieties. We previously demonstrated that mice exert very strong preferences when given binary mixtures of wheat varieties. We plan to utilize mice to identify wheat genes associated with flavor, and then relate this back to human preferences. Here we explore the effects of experimental design including the number of days (from 1 to 4) and number of mice (from 2 to 15) in order to identify designs that provide significant statistical inferences while minimizing requirements for labor and animals. When mice expressed a significant preference between 2 wheat varieties, increasing the number of days (for a given number of mice) increased the significance level (decreased P-values) for their preference, as expected, but with diminishing benefit as more days were added. However, increasing the number of mice (for a given number of days) provided a more dramatic log-linear decrease in P-values and thus increased statistical power. In conclusion, when evaluating mouse feeding preferences in binary mixtures of grain, an efficient experimental design would emphasize fewer days rather than fewer animals thus shortening the experiment duration and reducing the overall requirement for labor and animals.

Phytochemical composition, anti-inflammatory, and antiproliferative activity of whole wheat flour.

Whole wheat flour from five wheat cultivars was evaluated for phenolic, carotenoid, and tocopherol compositions as well as anti-inflammatory and antiproliferative activities against HT-29 cells. The total ferulic acid content ranged from 452 to 731 µg/g among the five cultivars and was primarily present in the insoluble-bound form. Lutein was the only carotenoid detected and ranged from 1.5 to 4.0 µg/g, and α-tocopherol levels ranged from 12 to 61 µg/g. Extracts of four cultivars demonstrated significant anti-inflammatory activity, measured as inhibition of interleukin-1ß (IL-1ß) mRNA expression; however, none of the extracts inhibited tumor necrosis factor-α (TNF-α) mRNA expression, a second indicator of anti-inflammatory activity. Proliferation of HT-29 adenocarcinoma cells was inhibited by extracts from all cultivars at the dose of 100 mg botanical equivalent/mL. The cultivar WestBred 936 had the greatest antiproliferative activity at lower concentrations (20 and 50 mg botanical equivalent/mL), had the greatest anti-inflammatory effect against IL-1ß, and also had the highest levels of ferulic acid and α-tocopherol. This research shows that whole wheat flours of these five cultivars varied significantly in their contents of phenolics, carotenoids, and α-tocopherol as well as in their anti-inflammatory and antiproliferative potentials, suggesting the possibility that wheat varieties can be selected based on potential health benefits.

Activation of polyphenol oxidase in dormant wild oat caryopses by a seed-decay isolate of Fusarium avenaceum.

Incubation of dormant wild oat (Avena fatua L., isoline M73) caryopses for 1-3 days with Fusarium avenaceum seed-decay isolate F.a.1 induced activity of the plant defense enzyme polyphenol oxidase (PPO). Both extracts and leachates obtained from F.a.1-treated caryopses had decreased abundance of an ∼57 kDa antigenic PPO and increased abundance of antigenic PPOs ranging from ∼52 to 14 kDa, as compared to extracts and leachates from untreated caryopsis. Leachates from caryopsis incubated for 2 days with F.a.1 also had 5.1- and 7.5-fold more total PPO activity/g fwt and specific activity, respectively. Fractionation of leachate proteins by ion-exchange chromatography associated the majority of PPO activity with an ∼36 kDa protein from untreated caryopses and ∼36, 25, and 24 kDa proteins from F.a.1-treated caryopses. Predicted peptide sequences obtained from high-performance liquid chromatography-tandem mass spectrometry analyses indicated that the ∼57 and 36 kDa wild oat proteins had a strong similarity to wheat PPO. However, the 25 and 24 kDa proteins were most similar to a Chitinase and oxalate oxidase, respectively. Our results indicate that F.a.1-induced activation of latent PPO in wild oat caryopsis likely involves a cleavage mechanism allowing activated PPOs to be readily mobilized into their surrounding environment.

Phenolic and short-chained aliphatic organic acid constituents of wild oat (Avena fatua L.) seeds.

The objective of this research was to identify and quantify the phenolic and short-chained aliphatic organic acids present in the seeds of three wild-type populations of wild oat and compare these results to the chemical composition of seeds from two commonly utilized wild oat isolines (M73 and SH430). Phenolic acids have been shown to serve as germination inhibitors, as well as protection for seeds from biotic and abiotic stress factors in other species, whereas aliphatic organic acids have been linked to germination traits and protection against pathogens. Wild oat populations were grown under a "common garden" environment to remove maternal variation, and the resulting seeds were extracted to remove the readily soluble and chemically bound phenolic and aliphatic organic acid components. Compounds were identified and quantified using gas chromatography-mass spectrometry. Ferulic and p-coumaric acid comprised 99% of the total phenolic acids present in the seeds, of which 91% were contained in the hulls and 98% were in the chemically bound forms. Smaller quantities of OH benzoic and vanillic acid were also detected. Soluble organic acids concentrations were higher in the M73 isoline compared to SH430, suggesting that these chemical constituents could be related to seed dormancy. Malic, succinic, fumaric and azelaic acid were the dominant aliphatic organic acids detected in all seed and chemical fractions.

Allelic variation of polyphenol oxidase (PPO) genes located on chromosomes 2A and 2D and development of functional markers for the PPO genes in common wheat.

Polyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products, especially Asian noodles. Characterization of PPO genes and the development of their functional markers are of great importance for marker-assisted selection in wheat breeding. In the present study, complete genomic DNA sequences of two PPO genes, one each located on chromosomes 2A and 2D and their allelic variants were characterized by means of in silico cloning and experimental validation. Sequences were aligned at both DNA and protein levels. Two haplotypes on chromosome 2D showed 95.2% sequence identity at the DNA level, indicating much more sequence diversity than those on chromosome 2A with 99.6% sequence identity. Both of the PPO genes on chromosomes 2A and 2D contain an open reading frame (ORF) of 1,731 bp, encoding a PPO precursor peptide of 577 amino acids with a predicted molecular mass of approximately 64 kD. Two complementary dominant STS markers, PPO16 and PPO29, were developed based on the PPO gene haplotypes located on chromosome 2D; they amplify a 713-bp fragment in cultivars with low PPO activity and a 490-bp fragment in those with high PPO activity, respectively. The two markers were mapped on chromosome 2DL using a doubled haploid population derived from the cross Zhongyou 9507/CA9632, and a set of nullisomic-tetrasomic lines and ditelosomic line 2DS of Chinese Spring. QTL analysis indicated that the PPO gene co-segregated with the two STS markers and was closely linked to SSR marker Xwmc41 on chromosome 2DL, explaining from 9.6 to 24.4% of the phenotypic variance for PPO activity across three environments. In order to simultaneously detect PPO loci on chromosomes 2A and 2D, a multiplexed marker combination PPO33/PPO16 was developed and yielded distinguishable DNA patterns in a number of cultivars. The STS marker PPO33 for the PPO gene on chromosome 2A was developed from the same gene sequences as PPO18 that we reported previously, and can amplify a 481-bp and a 290-bp fragment from cultivars with low and high PPO activity, respectively. A total of 217 Chinese wheat cultivars and advanced lines were used to validate the association between the polymorphic fragments and grain PPO activity. The results showed that the marker combination PPO33/PPO16 is efficient and reliable for evaluating PPO activity and can be used in wheat breeding programs aimed for noodle and other end product quality improvement.

Spatial distribution and differential expression of the PBAN receptor in tissues of adult Helicoverpa spp. (Lepidoptera: Noctuidae).

Pheromone-biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone production in many female moths. PBAN-like peptides, with common FXPRLamide C-terminals are found in other insect groups where they have other functions. The ubiquity and multifunctional nature of the pyrokinin/PBAN family of peptides suggests that the PBAN receptor proteins could also be present in a variety of insect tissues with alternative functions from that of sex pheromone biosynthesis. Previously we showed the presence of the PBAN-R in Helicoverpa armigera at the protein level. In the present study we confirm the similarities between the two Helicoverpa species: armigera and zea by (1) demonstrating the presence of the receptor protein in Sf9 cells, cloned to express the HezPBAN receptor, as compared with the endogenous receptor protein, previously shown in H. armigera pheromone glands, and (2) by identifying the nucleotide sequence of the PBAN-R from mRNA of H. armigera pheromone glands. Sequences of the two Helicoverpa spp. are 98% identical with most changes taking place in the 3'-end. We demonstrate the spatial distribution of the PBAN receptor protein in membranes of H. armigera brain (Br), thoracic ganglion (TG) and ventral nerve cord (VNC). We also demonstrate the presence and differential expression of the PBAN receptor gene (using reverse transcription-polymerase chain reaction and reverse transcription-quantitative real-time polymerase chain reaction, respectively) in the neural tissues (Br, TG and VNC) of adult H. armigera female moths as compared with its presence in pheromone glands. Surprisingly, the gene for the PBAN receptor is also detected in the male tissue homologous to the female pheromone gland, the aedeagus, although the protein is undetectable and PBAN does not induce physiological (pheromone production) or cellular (cyclic-adenosine monophosphate production) responses in this tissue. Our findings indicate that PBAN or PBAN-like receptors are present in the neural tissues and may represent a neurotransmitter-like function for PBAN-like peptides. In addition, the surprising discovery of the presence of the gene encoding the PBAN receptor in the male homologous tissue, but its absence at the protein level, launches opportunities for studying molecular regulation pathways and the evolution of these G protein coupled receptors (GPCRs).

Delineating the role of polyphenol oxidase in the darkening of alkaline wheat noodles.

This study evaluated the effects of inhibitors on polyphenol oxidase (PPO) activity, the effect of the PPO inhibitor tropolone on noodle darkening, and the correlation of PPO activity with darkening of alkaline noodles. The PPO inhibitors tropolone and salicylhydroxamic acid (each at 1 microM) reduced kernel PPO activity by approximately 50% in three hexaploid wheat cultivars but did not inhibit PPO activity in the two very low PPO cultivars, durum Langdon, and the synthetic hexaploid-derived ID580. Tropolone (100 microg/g flour) inhibited alkaline noodle darkening (deltaL*) by 13-25% in the low PPO wheat cultivar, ID377s, and by 39-54% in the high PPO wheat cultivar, Klasic. Alkaline noodle darkening among 502 wheat samples was correlated with kernel PPO activity (r = 0.64). Results substantiate the hypothesis that PPO plays a major role in darkening of alkaline noodles. However, results also indicate that substantial darkening would occur even at zero PPO activity, as measured in the kernel PPO assay. Therefore, darkening of alkaline noodles is probably due to the cultivar-specific level of PPO activity and the presence of at least one additional darkening mechanism. Further investigation is required to identify the phenolic discoloration agent(s) and to determine the potential roles of non-PPO discoloration mechanisms, both enzymatic and nonenzymatic, in wheat products.

Half-embryo cocultivation technique for estimating the susceptibility of pea (Pisum sativum L.) and lentil (Lens culinaris Medik.) cultivars to Agrobacterium tumefaciens.

Longitudinally sliced embryonic axes from pea and lentil mature seeds cocultivated with A. tumefaciens carrying a gus reporter gene in its T-DNA provided a convenient means to evaluate the efficiency of gene transfer to tissues in different cultivars and cocultivation conditions. Use of this technique demonstrated wide variation in susceptibility to Agrobacterium among several pea and lentil commercial genotypes.

Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes.