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Functionality of endogenous folates from rye and orange juice using human in vivo model.

BACKGROUND: Cereals contribute about a quarter of the daily folate intake from a typical diet in several European countries. However, studies on bioavailability of endogenous folates, in particular of cereal sources, are scarce. AIM OF THE STUDY: We aimed to study how well natural folates from rye (different rye breads and muesli made of malted rye) and orange juice function in improving folate status of human volunteers compared to a diet containing folic acid fortified wheat bread. METHODS: Healthy human volunteers aged 20-66 y took part in a four-week intervention trial in which bread, breakfast cereal and juice were provided. The study had a parallel design with two groups, 1) rye and orange juice group (33 volunteers) and 2) fortified wheat bread and apple juice group (31 volunteers). The test foods provided on average 184 microg and 188 microg folate per day in rye and wheat groups, respectively. Test foods were consumed as part of the subjects' normal diet. RESULTS: In both groups statistically significant increases in serum and red cell folates were observed after the intervention period. The serum folate increased 26 % and 31 %, and red cell folate levels increased 17 % and 15 % in rye and orange juice and wheat and apple juice groups, respectively. The effects did not differ between the rye and wheat groups. Increases in serum and red cell folate were more profound among subjects with low starting folate levels. Decrease in the plasma homocysteine concentrations was observed only in the highest tertile of both groups but not in the group means. CONCLUSIONS: Endogenous folates incorporated into a healthy diet, even in moderate amounts, is an efficient way to improve folate status among healthy adults. Folates from different rye products and orange juice showed good bioavailability that was similar to folic acid from fortified white bread.

Adult↗

Rye bran bread intake elevates urinary excretion of ferulic acid in humans, but does not affect the susceptibility of LDL to oxidation ex vivo.

BACKGROUND: Rye bread contributes an important part of the whole grain intake in the Scandinavian diet. Ferulic acid is the major phenolic compound in rye bran and is an antioxidant in vitro and may, therefore, contribute to cardioprotective effects of whole grain consumption. AIM OF THE STUDY: Firstly, to evaluate the bioavailability and potential antioxidative effects in humans of ferulic acid from rye. Secondly, to evaluate urine levels of ferulic acid as a possible biomarker of the ordinary dietary intake of ferulic acid. METHODS: We determined the urinary excretion of ferulic acid in 18 postmenopausal women after a dietary intake of rye bran or an inert wheat bran (control) in a crossover study (2 x 6 weeks with 4 weeks washout). The potential antioxidative effect of the rye bran intervention was investigated by measuring low-density lipoprotein (LDL) susceptibility to copper oxidation ex vivo. The subjects ingested rye bran enriched breads equivalent to approximately 10.2 mg ferulic acid per day. RESULTS: The urinary excretion of ferulic acid averaged approximately 4.8 mg per day during intervention with rye bran breads and approximately 1.9 mg per day on the control breads (P = 0.002). Rye bran intervention had no influence on lag time or propagation rate of the LDL oxidation ex vivo. CONCLUSIONS: The present study demonstrated that ferulic acid from rye bran is bioavailable and that the urinary concentration of ferulic acid reflects the dietary intake of this hydroxycinnamic acid. Within the period of intervention, the elevated ferulic acid did not produce a measurable antioxidative effect on the subjects' LDL. It is suggested that the determination of ferulic acid in urine is a useful biomarker to assess the intake of ferulic acid from a regular diet.

Aged↗

Suitability of a batch in vitro fermentation model using human faecal microbiota for prediction of conversion of flaxseed lignans to enterolactone with reference to an in vivo rat model.

BACKGROUND: In vitro fermentation models have been used widely for studies of shortchain fatty acid (SCFA) formation from carbohydrates, whereas the suitability of these methods for enterolactone (ENL) formation has received less attention. AIM: The aim was to study the suitability of an in vitro fermentation model for prediction of bioconversion of lignans to ENL, to compare the approach with that of an in vivo rat model and to study the SCFA formation in both models. METHODS: Predigested samples of rye bran (R), flaxseed meal (F) alone, or in combination with rye bran (R&F) and a faecal control were incubated in an in vitro fermentation model using human faecal microbiota. In the in vivo experiment rats consumed a non-fibre control diet (C) or diets supplemented either with rye bran (R), flaxseed meal (F) alone, or with their combination (R&F) for four weeks. Enterodiol (END), ENL and SCFA concentrations were measured from in vitro faecal fermentation samples and from the intestinal contents of rats. Plasma ENL concentrations from rats were also measured. RESULTS: The highest ENL production was found in vitro with the F supplement (areas under curve: 740 +/- 4, 7,500 +/- 400, 2,600 +/- 500 and 1,520 +/- 70 nmol x h for the R, F, R&F supplements and faecal control, respectively). In vivo, the concentration of ENL in caecal digesta from flaxseed meal was significantly (P < 0.05) enhanced by the presence of rye bran (medians 261, 407 and 24 nmol/g in the F, R&F and C groups, respectively). No correlation was found between the models regarding ENL production, possibly due to different responses to the presence of rye bran matrix, differences in microbiota or application of a batch in the in vitro fermentation model. Rye bran supplementation enhanced butyrate production both in vitro and in vivo. CONCLUSION: In vitro fermentation and the in vivo rat models responded differently to the presence of rye bran and no correlation with regard to the ENL formation from flaxseed lignans was observed.

4-Butyrolactone↗

Rye terminal neocentromeres: characterisation of the underlying DNA and chromatin structure.

We have studied rye plants with neocentromeres on the terminal regions of the chromosomes. These neocentromeres only appear in meiosis, they are active together with the normal centromere and move the chromosomal arms polewards from prometaphase to anaphase at both the first and second meiotic divisions. All chromosomes of the normal set may show neocentric activity, but chromosomal arms with terminal heterochromatic blocks, as assessed by C-banding, are significantly more susceptible than those that do not have them. At least three repetitive sequences underlie the neocentromeres: pSc34, pSc74 and pSc200. These sequences are not detectable in B chromosomes, which never showed neocentric activity. Fluorescence in situ hybridisation with these sequences used as probes revealed elongated chromatin extensions on the neocentromeres that have not been observed using other staining techniques. These extensions were never observed in control plants. They suggest a modified chromatin structure, which might be responsible for the interaction with proteins involved in chromosomal movement on the spindle.

Cell Nucleus↗

Diverse patterns of the tandem repeats organization in rye chromosomes.

Although the monomer size, nucleotide sequence, abundance and species distribution of tandemly organized DNA families are well characterized, little is known about the internal structure of tandem arrays, including total arrays size and the pattern of monomers distribution. Using our rye specific probes, pSc200 and pSc250, we addressed these issues for telomere associated rye heterochromatin where these families are very abundant. Fluorescence in situ hybridization (FISH) on meiotic chromosomes revealed a specific mosaic arrangement of domains for each chromosome arm where either pSc200 or pSc250 predominates without any obvious tendency in order and size of domains. DNA of rye-wheat monosomic additions studied by pulse field gel electrophoresis produced a unique overall blot hybridization display for each of the rye chromosomes. The FISH signals on DNA fibres showed multiple monomer arrangement patterns of both repetitive families as well as of the Arabidopsis-type telomere repeat. The majority of the arrays consisted of the monomers of both families in different patterns separated by spacers. The primary structure of some spacer sequences revealed scrambled regions of similarity to various known repetitive elements. This level of complexity in the long-range organization of tandem arrays has not been previously reported for any plant species. The various patterns of internal structure of the tandem arrays are likely to have resulted from evolutionary interplay, array homogenization and the generation of heterogeneity mediated by double-strand breaks and associated repair mechanisms.

Chromosomes, Plant↗

Centromere association is an unlikely mechanism by which the wheat Ph1 locus regulates metaphase I chromosome pairing between homoeologous chromosomes.

Chiasmate pairing between homoeologous chromosomes at metaphase I (MI) of meiosis in wheat is prevented by the activity of the Ph1 locus on chromosome 5B. Several hypotheses have been proposed sharing the assumption that Ph1 regulates MI chromosome pairing by regulating centromere-mediated chromosome alignment before the onset of meiosis. To test the relevance of the putative predetermination of chromosome pairing at MI by the centromere-mediated chromosome association prior to meiosis, a 2BL.2RL homoeoisochromosome was constructed and its MI pairing was assessed in the presence and absence of the Ph1 locus. Although the 2BL and 2RL arms of the homoeoisochromosome paired with each other at MI in the absence of Ph1, they never paired with each other at MI in the presence of Ph1. Since the two arms were permanently associated in the homoeoisochromosome via a common centromere, it is unlikely that Ph1 predetermines MI pairing between homoeologous chromosomes solely by controlling premeiotic association of centromeres. These findings are consistent with the idea that Ph1 determines the chromosome pairing pattern at MI by scrutinizing homology across the entire chromosome.

Centromere↗

Centromeric behaviour in wheat with high and low homoeologous chromosomal pairing.

Control of homoeologous chromosomal pairing in hexaploid wheat stems from a balance between a number of suppressor and promoter genes. This study used centromeric behaviour as a tool to investigate the mechanism. Fluorescent in situ hybridization employing centromeric and telomeric sequences as probes was applied to pollen mother cells of wheat and wheat/alien hybrids having different pairing gene combinations. It showed: association of centromeres during pre-meiotic interphase; decondensation of centromeric structure; sister chromatid disjunction of univalent chromosomes in homoeologous pairing situations at anaphase I; and centromeric stretching between univalent sister chromatids in wheat/rye hybrids deficient for pairing genes. The implications of these results are discussed.

Anaphase↗

The effect of the wheat Ph1 locus on chromatin organisation and meiotic chromosome pairing analysed by genome painting.

The Ph1 locus in wheat influences homo(eo)logous chromosome pairing. We have analysed its effect on the behaviour and morphology of two 5RL rye telosomes in a wheat background, by genomic in situ hybridisation (GISH), using rye genomic DNA as a probe. Our main objective was to study the effect of different alleles of the Ph1 locus on the morphology and behaviour of the rye telosomes in interphase nuclei of tapetal cells and in pollen mother cells at early stages of meiosis. The telosomes, easily detectable at all stages, showed a brightly fluorescing chromomere in the distal region and a constriction in the proximal part. These diagnostic markers enabled us to define the centromere and telomere regions of the rye telosomes. In the presence of functional copies of Ph1, the rye telosomes associated at pre-leptotene, disjoined and reorganised their shape at leptotene, and became fully homologously paired at zygotene - pachytene. In plants without functional alleles (ph1bph1b), the rye telosomes displayed an aberrant morphology, their premeiotic associations were clearly disturbed and their pairing during zygotene and pachytene was reduced and irregular. The Ph1 locus also influenced the behaviour of rye telosomes in the interphase nuclei of tapetal cells: in Ph1Ph1 plants, the rye telosomes occupied distinct, parallel-oriented domains, whereas in tapetal nuclei of ph1bph1b plants they were intermingled with wheat chromosomes and showed a heavily distorted morphology. The results shed new light on the effect of Ph1, and suggest that this locus is involved in chromosome condensation and/or scaffold organisation. Our explanation might account for various apparently contradictory and pleiotropic effects of this locus on both premeiotic associations of homologues, the regulation of meiotic homo(eo)logous chromosome pairing and synapsis, the resolution of bivalent interlockings and centromere behaviour.

Cell Nucleus↗

The chromosomal organization of simple sequence repeats in wheat and rye genomes.

The physical distribution of ten simple-sequence repeated DNA motifs (SSRs) was studied on chromosomes of bread wheat, rye and hexaploid triticale. Oligomers with repeated di-, tri- or tetra-nucleotide motifs were used as probes for fluorescence in situ hybridization to root-tip metaphase and anther pachytene chromosomes. All motifs showed dispersed hybridization signals of varying strengths on all chromosomes. In addition, the motifs (AG)12, (CAT)5, (AAG)5, (GCC)5 and, in particular, (GACA)4 hybridized strongly to pericentromeric and multiple intercalary sites on the B genome chromosomes and on chromosome 4A of wheat, giving diagnostic patterns that resembled N-banding. In rye, all chromosomes showed strong hybridization of (GACA)4 at many intercalary sites that did not correspond to any other known banding pattern, but allowed identification of all R genome chromosome arms. Overall, SSR hybridization signals were found in related chromosome positions independently of the motif used and showed remarkably similar distribution patterns in wheat and rye, indicating the special role of SSRs in chromosome organization as a possible ancient genomic component of the tribe Triticeae (Gramineae).

Base Sequence↗

The centromere structure in Robertsonian wheat-rye translocation chromosomes indicates that centric breakage-fusion can occur at different positions within the primary constriction.

Univalent chromosomes at meiotic metaphase I have a tendency to misdivide at the centromeres. Fusion of the misdivision products may produce Robertsonian translocations. The fine structure of the centromeres in Robertsonian wheat-rye translocation chromosomes was analyzed by fluorescence in situ hybridization (FISH) using two centromere-specific DNA clones: pRCS1, derived from rice, and pAWRC1, derived from rye. Clone pRCS1 hybridizes to the centromeres of all grasses including wheat and rye, whereas clone pAWRC1 is rye specific and hybridizes only to the centromeres of rye. Four of the six wheat-rye translocations derived from a single centric misdivision event (1st generation translocations) had hybrid centromeres, with approximately half of the centromere derived from rye and half from wheat. In the two other 1st generation translocations, the entire centromere was derived from rye. Among eight reconstructed wheat and rye chromosomes that originated from two consecutive centric misdivision-fusion events (2nd generation translocations), T1BS.1BL (derived from T1BS.1RL and T1RS.1BL) and one of three T2BS.2BL (derived from T2RS.2BL and T2BS.2RL) had hybrid centromeres. T1RS.1RL (derived from T1BS.1RL and T1RS.1BL), two of three T2BS.2BL, and all three T2RS.2RL (derived from T2RS.2BL and T2BS.2RL) had rye centromeres. All three 3rd generation translocations had hybrid centromeres with approximately half of the centromere derived from rye. There were no indications that the composite structure of the centromere in these chromosomes affected their behavior in mitosis or meiosis. These observations support the notion of a compound structure of the centromere in higher organisms, and indicate that during the centric breakage-fusion event, centromere breakage may occur in different positions along the segment of the chromosome that interacts with the spindle fibers. Normal behavior of the 1st, 2nd, and 3rd generation centric translocations in mitosis and meiosis indicates that, at least in wheat and rye, centromeres are not chromosome specific.

Cell Line↗

Cytosine methylation and nucleolar dominance in cereal hybrids.

In wheat-rye hybrids the nucleolus organizer regions (NORs), the sites of ribosomal RNA genes, from rye are suppressed. Wheat and wheat-rye hybrid genetic stocks containing different numbers of wheat and rye nucleolus organizers, as well as addition lines and rye-barley hybrids, were used in Southern hybridization experiments to determine the cause of nucleolar dominance and suppression in cereal hybrids. Based on the use of restriction endonucleases that cleave near the ends of the spacer unit and an additional, methylation-sensitive enzyme, HpaII, which does not recognize the CCGG restriction site if the internal C is methylated, an indirect method of assaying NOR expression was established. The results indicated that cleavage by the HpaII enzyme of the rye NOR sequences, is reduced when major NORs from other cereals were present. The reduction in the number of rye rRNA genes containing an unmethylated CCGG site in the promoter was associated with the suppression of the rye nucleolus. These results are consistent with a model in which promoter and upstream regulatory repeats of ribosomal RNA genes compete for limited concentrations of regulatory proteins, and genes that are methylated at key binding sites fail to engage these regulatory proteins and thus remain inactive.

Cell Nucleolus↗

Isolation of lambda and YAC clones from defined regions of the rye genome.

A dispersed, rye-specific element has been used to isolate clones of rye origin from wheat plants containing only a single rye chromosome arm or segment. In this way a set of 23 YAC clones has been isolated from the short arm of rye chromosome 1 (1RS). This technique was extended to isolate clones from a small region of 1RS that contains a large number of agronomically important genes. The targeted cloning method allowed the isolation of 26 classes of lambda clones representing about 5% of the region. Ten of the lambda clones could be mapped to segments within this region. A third example of the application of this technique involved the isolation of clones from a very small but fully functional rye chromosome, the midget chromosome. These clones have allowed the confirmation of the origin of the midget from 1RL, and may provide a tool for the isolation of structural elements of cereal chromosomes. This technique allows the identification of clone libraries for any rye chromosome or chromosome arm, since substitution, addition and translocation lines are available for all rye chromosomes. Furthermore, the technique allows isolation of clones derived from segments of the rye genome recombined into wheat. The method is technically simple and both lambda and YAC libraries can be constructed. Synteny between the genomes of the cereals allows region-specific libraries from rye to be used to target regions of the wheat and barley genomes.

Bacteriophage lambda↗

Molecular characterization of a new furovirus mainly infecting rye.

The complete or almost complete nucleotide sequences were determined for the two RNAs of three different sources of a new furovirus which mainly infects rye and for which the name Soil-borne rye mosaic furovirus (SBRMV) is proposed. The genome organization of this virus is virtually identical to that of Soil-borne wheat mosaic furovirus (SBWMV). However, SBRMV and SBWMV differ considerably in the nucleotide sequences of their genomes and in the derived amino acid sequences of their putative gene products. The sequences of the three sources of SBRMV (two from rye and one from wheat) differ between each other in various parts of their genomes by 1% to 10%. Larger differences were found in the readthrough part of the coat protein readthrough protein. There are no indications that the relationship of the C source from wheat to the other two sources from rye is more distant than the relationship between the two sources from rye. The 3'UTRs of both SBRMV RNAs are remarkable in having a predicted upstream pseudoknot domain (UPD) with seven pseudoknots. The same number was also predicted for the UPD of SBWMV RNA 1 whereas the much shorter UDP of SBWMV RNA 2 may form only four pseudoknots.

3' Untranslated Regions↗

Soil-borne rye mosaic and European wheat mosaic virus: two names for a furovirus with variable genome properties which is widely distributed in several cereal crops in Europe.

PCR and nucleotide sequence analyses have revealed that Soil-borne rye mosaic furovirus (SBRMV) which we have recently described is widely distributed in Europe. In Northern Germany, Poland and Denmark the virus affects mainly rye and triticale, but in France and Italy it is wheat which becomes infected. The partial RNA 1 and RNA 2 sequences which were determined for the various SBRMV sources form several clusters, but so far no correlation between molecular differences and the type of host which becomes infected under natural conditions was detected. European wheat mosaic furovirus which was recently described by Diao et al. (1999) [Virology 261: 331-339] is closely related to a French source of SBRMV.

Base Sequence↗

Gravitropic plant growth regulation and ethylene: an unsought cardinal coordinate for a disused model.

According to the Cholodny-Went hypothesis, gravitropic differential growth is brought about by the redistribution of auxin (indolyl-3-acetic acid, IAA). We reinvestigated the relevance of different auxins and studied the role of ethylene in hypocotyls of sunflower and shoots and roots of rye and maize seedlings. Incubation of coleoptiles and of sunflower hypocotyls in solutions of IAA and dichlorophenoxyacetic acid as well as naphthylacetic acid resulted in a two- to threefold length increase compared to water controls. In spite of this pronounced general effect on elongation growth, gravi-curvature was similar to water controls. In contrast to this, inhibition of ethylene synthesis by aminoethoxyvinylglycine prevented differential growth of both hypocotyls and coleoptiles and of roots of maize. In horizontally stimulated maize roots growing on surfaces, inhibition of ethylene perception by methylcyclopropene inhibited roots to adapt growth to the surface, resulting in a lasting vertical orientation of the root tips. This effect is accompanied by up- and down-regulation of a number of proteins as detected by two-dimensional matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Together the data query the regulatory relevance of IAA redistribution for gravitropic differential growth. They corroborate the crucial regulatory role of ethylene for gravitropic differential growth, both in roots and coleoptiles of maize as well as in hypocotyls.

2,4-Dichlorophenoxyacetic Acid↗

Identification and physical mapping of induced translocation breakpoints involving chromosome 1R in rye.

To obtain translocations involving specific chromosomes in rye, pollen of a line in which chromosome 1R has large C-bands on its two telomeres, but which lacks C-bands (or has very small ones) on the telomeres of the remaining chromosomes, was X-irradiated. All translocations involving the labelled chromosome (1R) could be easily recognized in C-banded mitotic metaphases. The non-labelled chromosome involved in each translocation was identified either from mitotic C-banding analysis or from the meiotic configurations observed in some specific progenies. A physical map including 40 translocation breakpoints has been developed by means of synaptonemal complex (SC) analysis of well-paired pachytene quadrivalents. The results agree with the hypothesis of chromosomes 2R to 7R having similar probabilities of participating in translocations with chromosome 1R. However, the locations of the breakpoints are not entirely random: an excess of translocation breakpoints located on the short arm of chromosome 1R was obtained, and the two acentric translocated segments of each translocation show a trend towards having similar sizes. The possible reasons for these two non-random situations are discussed.

Chromosome Banding↗

Genic heterozygosity, chromosomal interchanges and fitness in rye: any relationship?

Relationship between heterozygosity at allozyme loci, chromosomal interchanges and fitness was analyzed in a rye cultivar showing a polymorphism for such rearrangements. Nine allozyme systems (ACO, ACPH, GOT, GPI, LAP, MDH, PER, PGD and PGM) and five components of fitness (number of fertile tillers, total offspring, egg cell fertility, flowers/ear and seeds/ear) were studied. The estimated selection coefficients against interchange heterozygotes ranged from s = 0.12 to s = 0.34. A significant effect of the genic heterozygosity on some fitness components was observed in interchange heterozygotes (tillering and total offspring), in their standard homozygous sibs (flowers/ear and seeds/ear) and in the descendants of the crosses between standard karyotypes (flowers/ear, seeds/ear and egg cell fertility). However, the main effect was linked to genetic background associated to different crosses. Significant differences for Acph-1, Gpi-1, Lap-1, Mdh-1, Mdh-4, Pgd-2 and Pgm-1 loci were also found in some of these crosses although these differences were inconsistent. This suggests that probably the allozyme loci analyzed were not directly contributing to the fitness and that they are linked, in some cases, to different deleterious alleles depending on both cross and locus. This fact could support the local effect hypothesis as explanation although we do not discard the existence of some inbreeding level (general effect hypothesis) since all crosses and loci studied show a overall consistent trend of increased fitness with increased heterozygosity.

Alleles↗

Biotransformation of 2-benzoxazolinone to 2-amino-(3H)-phenoxazin-3-one and 2-acetylamino-(3H)-phenoxazin-3-one in soil.

An alternative to the use of synthetic pesticides is to exploit the natural defense chemicals produced by cereals. An important class of allelochemicals is cyclic hydroxamic acids and related benzoxazolinones. A prolonged degradation experiment of the allelochemical compound from rye 2-benzoxazolinone (BOA) was carried out for up to 90 d at 15 degrees C at three different concentration levels, 3, 3000, and 30,000 nmol BOA g soil(-1), respectively, in a sandy loam soil. Two main degradation products, 2-amino-(3H)-phenoxazin-3-one (APO) and 2-acetylamino-(3H)-phenoxazin-3-one (AAPO), were identified and quantified by LC-ESI-MS-MS. The half-life of BOA increased with higher levels of BOA added to the soil. Half-lives of BOA, APO, and AAPO were determined by fitting a single first-order model to the degradation data. Half-life of BOA was determined to be 0.6 d in the 3 nmol BOA g soil(-1) treatment. Half-lives of BOA, APO, and AAPO were 3.1, 2.7, and 2.1 d, respectively, in the 3000 nmol BOA g soil(-1) treatment. In the 30,000 nmol BOA g soil(-1) treatment, the half-lives were 31 d for BOA and 45 d for APO. The microbial community structure was not affected by addition of BOA to the soil as investigated by analysis of signature fatty acids. The results suggest that the exploitability of BOA for crop protection is dependent on the existing concentration of BOA in the soil and the timing of incorporation of hydroxamic acid synthesizing crops into the soil.

Agriculture↗