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Characterization and genomic organization of Ty1-copia group retrotransposons in rye (Secale cereale).

The genomic organisation of the Ty1-copia retrotransposons in rye (Secale cereale) has been studied. We have used the polymerase chain reaction (PCR) to amplify sequences from a conserved domain of the reverse transcriptase gene of the Ty1-copia retrotransposons in this species. Sequence analysis of 26 of these PCR products shows them to be a highly heterogeneous population, a feature that is common in plants. Slot blot analysis shows that there are about 100,000 individual Ty1-copia retrotransposons in rye. In situ hybridization of a heterogeneous probe, representing the whole population of rye Ty1-copia retrotransposon sequences, to chromosome spreads of triticale (xTriticosecale), a rye-wheat hybrid, shows that these sequences are present throughout all the rye chromosomes but absent from the centromeric regions and, in particular, from the terminal heterochromatin. Southern analysis of oat, barley, wheat, and rye, using as a probe R9, one of the rye sequences that is closely similar to the BARE-1 element of barley, shows that close relatives of this retrotransposon subgroup are present in all these species in high copy number. Northern analysis on RNAs from seedlings shows that the BARE-1 subgroup is transcribed in all these cereal plants but in variable amounts: high in barley, moderate in wheat and rye, and extremely low in oat.

Amino Acid Sequence↗

Chromosome condensation in mitosis and meiosis of rye (Secale cereale L.).

Structural investigation and morphometry of meiotic chromosomes by scanning electron microscopy (in comparison to light microscopy) of all stages of condensation of meiosis I + II show remarkable differences during chromosome condensation in mitosis and meiosis I of rye (Secale cereale) with respect to initiation, mode and degree of condensation. Mitotic chromosomes condense in a linear fashion, shorten in length and increase moderately in diameter. In contrast, in meiosis I, condensation of chromosomes in length and diameter is a sigmoidal process with a retardation in zygotene and pachytene and an acceleration from diplotene to diakinesis. The basic structural components of mitotic chromosomes of rye are "parallel fibers" and "chromomeres" which become highly compacted in metaphase. Although chromosome architecture in early prophase of meiosis seems similar to mitosis in principle, there is no equivalent stage during transition to metaphase I when chromosomes condense to a much higher degree and show a characteristic "smooth" surface. No indication was found for helical winding of chromosomes either in mitosis or in meiosis. Based on measurements, we propose a mechanism for chromosome dynamics in mitosis and meiosis, which involves three individual processes: (i) aggregation of chromatin subdomains into a chromosome filament, (ii) condensation in length, which involves a progressive increase in diameter and (iii) separation of chromatids.

Chromosomes, Plant↗

Ultrastructural analysis of chromatin in meiosis I + II of rye (Secale cereale L.).

Scanning electron microscopy (SEM) proves to be an appropriate technique for imaging chromatin organization in meiosis I and II of rye (Secale cereale) down to a resolution of a few nanometers. It could be shown for the first time that organization of basic structural elements (coiled and parallel fibers, chromomeres) changes dramatically during the progression to metaphase I and II. Controlled loosening with proteinase K (after fixation with glutaraldehyde) provides an enhanced insight into chromosome architecture even of highly condensed stages of meiosis. By selective staining with platinum blue, DNA content and distribution can be visualized within compact chromosomes as well as in a complex arrangement of fibers. Chromatin interconnecting threads, which are typically observed in prophase I between homologous and non-homologous chromosomes, stain clearly for DNA. In zygotene transversion of chromatid strands to their homologous counterparts becomes evident. In pachytene segments of synapsed and non-synapsed homologs alternate. At synapsed regions pairing is so intimate that homologous chromosomes form one filament of structural entity. Chiasmata are characterized by chromatid strands which traverse from one homolog to its counterpart. Bivalents are characteristically fused at their telomeric regions. In metaphase I and II there is no structural evidence for primary and secondary constrictions.

Centromere↗

Meiotic mutations in rye Secale cereale L.

Spontaneous meiotic mutations of winter rye Secale cereale L. (2n = 14) were revealed in inbred F2 progenies, which were obtained by self-pollination of F1 hybrids resulting from crosses of individual plants of cultivar Vyatka or weedy rye with plants of self-fertile inbred lines. The mutations cause partial or complete sterility, and are maintained in heterozygote condition. Six types of mutations were distinguished as the result of cytological analysis of meiosis and genetic analysis. (1) Plants with nonallelic asynaptic mutations sy1 and sy9 lacked bivalents in 96.8 and 67.0% metaphase I cells, respectively, formed only axial elements but not the mature synaptonemal complex (SC), and had defects in telomere clustering in early prophase I. (2) Weak asynaptic mutant sy3 showed incomplete synapsis at the start of SC degradation at diplotene and lower chiasma number; yet only 2% meiocytes lacked bivalents in MI. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19 caused nonhomologous synapsis; i.e., a varying number of univalents and occasional multivalents were observed in MI, which was preceded by switches of pairing partners and fold-back synapsis at mid-prophase I. (4) Mutation mei6 led to the formation of protrusions and minor branched structures of the SC lateral elements. (5) Allelic mutations mei8 and mei8-10 caused irregular chromatin condensation along the chromosome length in prophase I, which was accompanied by chromosome sticking and fragmentation in MI. (6) Allelic mutations mei5 and mei10 determined chromosome supercondensation, caused the disturbance of meiotic spindle assembly, arrested meiosis at various stages but did not affect formation of the pollen wall, thus arrested meiocytes got covered with the pollen wall. Analysis of double mutants revealed recessive epistatic interactions for some mutations; the epistatic group was sy9 > sy1 > sy3 > sy19. This reflects the sequence of meiotic events controlled by the corresponding genes. The expression of sy2 and sy19 proved to be modified by additional genes. Most meiotic mutations found in rye have analogs in other plants.

Chromosome Pairing↗

Analysis of rye pollen (Secale cereale) allergens using patients' IgE, immunoprint, Western Blot and monoclonal antibodies.

Rye pollen (Secale cereale) was investigated by Immunoprint and Western Blot analysis. Using Immunoprint, more than 30 proteins could be shown to be allergens in terms of their IgE binding, and 9 were determined as major allergens. In Western Blot 17 allergens were discovered, 3 of them major allergens. 4 monoclonal antibodies raised against a pollen extract of 6 grasses showed cross-reactivity to rye pollen, thus indicating common epitopes. 1 of 3 monoclonal antibodies produced against an extract of rye pollen was only reacting to this pollen. Looking at the overall binding pattern of the 7 monoclonal antibodies, they showed partial identity to patients' IgE in Immunoprint and Western Blot. The possibility of defining epitopes using preparative isoelectric focusing and monoclonal antibodies is discussed.

Antibodies, Monoclonal↗

Nuclear dispositions of subtelomeric and pericentromeric chromosomal domains during meiosis in asynaptic mutants of rye (Secale cereale L.).

The nuclear dispositions of subtelomeric and pericentromeric domains in pollen mother cells (PMCs) were tracked during meiosis in wildtype and two asynaptic mutants of rye (Secale cereale L.) by means of fluorescence in situ hybridization (FISH). Homozygotes for sy1 and sy9 non-allelic mutations form axial elements during leptotene of male meiosis, but fail to form synaptonemal complexes. Consequently, recombination is severely impaired, and high univalency is observed at metaphase I. Simultaneous FISH with pSc200 subtelomeric tandem repeat and CCS1 centromeric sequence revealed that at pre-meiotic interphase the two domains are in a bipolar Rabl orientation in both the PMCs and tapetal cells. At the onset of meiotic prophase, the subtelomeric regions in PMCs of wildtype and sy9 cluster into a typical bouquet conformation. The timing of this event in rye is comparable with that in wheat, and is earlier than that observed in other organisms, such as maize, yeast and mammals. This arrangement is retained until later in leptotene and zygotene when the pericentromeric domains disperse and the subtelomeric clusters fragment. The mutant phenotype of sy9 manifests itself during leptotene to zygotene, when the pericentromeric regions become distinctly more distended than in wildtype, and largely fail to pair during zygotene. This indicates that difference in the nature or timing of chromosome condensation in this region is the cause or consequence of asynapsis. By contrast, sy1 fails to form comparable aggregates of subtelomeric regions at leptotene in only half of the nuclei studied. Instead, two to five aggregates are formed that fail to disperse at later stages of meiotic prophase. In addition, the pericentromeric regions disperse prematurely at leptotene and do not associate in pairs at any subsequent stage. It is supposed that the sy1 mutation could disrupt the nuclear disposition of centromeres and telomeres at the end of pre-meiotic interphase, which could cause, or contribute to, its asynaptic phenotype.

Cell Nucleus↗

Homolog pairing and two kinds of bouquets in the meiotic prophase of rye, Secale cereale.

Chromosome configurations and structures during meiotic prophase were investigated by staining large repeated DNA sequences localized in the subtelomeric regions of all the chromosomes in rye, Secale cereale, in order to clarify when and how homolog pairing and bouquet formation occur. The changes of the spatial locations of chromosomes in the nucleus were investigated by the use of laser confocal microscopy, together with the surface-spreading method of silver nitrate staining to detect the formation of the synaptonemal complex. Homolog pairing in which homologs of four chromatids of a pair of homologs were coaligned in parallel but remained distinctly separate was microscopically detected for the first time in the present study. Homolog pairing showed the following characteristics: (1) it occurred at the leptotene-zygotene transition stage, prior to the formation of nodules and the synaptonemal complex; (2) the chromatin structure of chromosomes was in a state of decondensation; (3) it required no telomere clustering. These data suggest that homolog pairing represents a structure that indicates incipient recombination. After the homolog pairing stage, two kinds of bouquet configuration were found in zygotene. The commonly observed type was a loose bouquet, in which the subtelomeric regions were loosely aggregated. The other type was a definite bouquet, in which almost all the subtelomeric regions were conjugated, but this type was observed only in a limited number of the meiotic prophase cells of some individuals. It was concluded that the former represents the configuration of homologous recombination and the latter that of ectopic recombination.

Cell Nucleus↗

Purification and some properties of three chitinases from the seeds of rye (Secale cereale).

Three chitinases, designated RSC-a, -b, and -c, were purified from the seeds of rye (Secale cereal) using ammonium sulfate precipitation, CM-cellulose column chromatography, gel filtration on Sephadex G-75, and S-Sepharose column chromatography. RSC-a, -b, and -c are basic proteins having molecular masses of 33 kDa, 26 kDa, and 26 kDa, and isoelectric points of 9.7, 10, and > 10, respectively. RSC-b and -c were found to be homologous proteins having similar amino acid compositions and N-terminal sequences. RSC-a contains more Thr, Ser, Glu, Pro, Gly, and Cys than RSC-b and -c and has a different N-terminal sequence from them. They hydrolyze glycolchitin and colloidal chitin, but not cell walls of Micrococcus lysodeikticus. These enzymes are stable at pH 4-8 and their optimum pHs toward glycolchitin are 5.

Amino Acid Sequence↗

Isolation and amino acid sequence of a protein-synthesis inhibitor from the seeds of rye (Secale cereale).

A protein-synthesis inhibitor, designated RPSI, was isolated from the seeds of rye (Secale cereale) using gel filtration and S-Sepharose column chromatography. RPSI is a basic protein with an isoelectric point of over 10, and the concentration of protein required for 50% inhibition of protein synthesis (IC50) of purified RPSI was about ten-fold the concentration of ricin A-chain. The complete amino acid sequence of RPSI was discovered by analyzing the peptides and fragments obtained from the proteolytic digests and by the cyanogen bromide- and hydroxylamine-cleavages of RPSI. RPSI consists of 280 amino acid residues and has a molecular weight of 30,171. RPSI has only 21% sequence identity with that of ricin A-chain, but all five amino acid residues involved in the active site of ricin A-chain are conserved in RPSI.

Amino Acid Sequence↗

Rye (Secale cereale L.).

Rye (Secale cereale L.) is one of the most recalcitrant plant species for tissue culture and genetic transformation. Embryogenic rye callus loses its ability to regenerate plants quickly in response to high density of Agrobacterium and other stressors. The cocultivation of Agrobacterium and rye immature embryos in liquid medium facilitated washing of the cultures to avoid Agrobacterium overgrowth and allowed a high throughput. More than 40 independent transgenic plants were regenerated with one to four Southern-positive, independent events from 100 inoculated immature embryos. Agrobacterium strain AGL0 supported stable integration of a constitutive nptII selectable marker expression cassette into the genome of rye inbred line L22, as indicated by regeneration of plantlets on paromomycin-containing culture medium, Southern blot, Western blot, and the analysis of T-DNA::plant DNA boundary sequences. Transgenic plants were phenotypically normal and fully fertile, which might be a consequence of the short time in tissue culture.

Agrobacterium tumefaciens↗

Hydroxamic acids in Secale cereale L. and the relationship with their antifeedant and allelopathic properties.

Contents of the hydroxamic acids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) in leaves and roots of 14 cultivars of rye, Secale cereale L., were determined. Dynamics of accumulation in three cultivars were evaluated. DIBOA was the main cyclic hydroxamic acid in leaves but the contents differed significantly between the cultivars. Both DIBOA and DIMBOA were present in the roots. Maximum concentration of DIBOA in leaves and DIMBOA in roots was reached between 48-54 h and 54-72 h after germination, respectively. Antifeedant activity of DIBOA towards the aphid Rhopalosiphum padi and the feeding behavior were studied by electronic recording in barley leaves treated with different contents of DIBOA. The deleterious activity of DIBOA could arise by starvation and/or a toxic effect. Additionally, allelopathic potential of pure DIBOA and aqueous extracts of leaves and roots of rye (Tetra-Baer) on the germination of lettuce (Lactuca sativa) and rye (Tetra-Baer) seeds was evaluated. A high percentage of germination inhibition of pure DIBOA and the extracts of leaves and roots was observed. The activity is in agreement with the contents of hydroxamic acids in the plants. The substrates had no allelopathic effect on rye seeds.

Animals↗

Molecular assembly of meiotic proteins Asy1 and Zyp1 and pairing promiscuity in rye (Secale cereale L.) and its synaptic mutant sy10.

Assembly of two orthologous proteins associated with meiotic chromosome axes in Arabidopsis thaliana (Asy1 and Zyp1) was studied immunologically at meiotic prophase of meiosis of wild-type rye (Secale cereale) and its synaptic mutant sy10, using antibodies derived from A. thaliana. The temporal and spatial expression of the two proteins were similar in wild-type rye, but with one notable difference. Unlike A. thaliana, in which foci of the transverse filament protein Zyp1 appear to linearize commensurately with synapsis, linear tracts of Asy1 and Zyp1 protein form independently at leptotene and early zygotene of rye and coalign into triple structures resembling synaptonemal complexes (SCs) only at later stages of synapsis. The sy10 mutant used in this study also forms spatially separate linear tracts of Asy1 and Zyp1 proteins at leptotene and early zygotene, and these coalign but do not form regular triple structures at midprophase. Electron microscopy of spread axial elements reveals extensive asynapsis with some exchanges of pairing partners. Indiscriminate SCs support nonhomologous chiasma formation at metaphase I, as revealed by multi-color fluorescence in situ hybridization enabling reliable identification of all the chromosomes of the complement. Scrutiny of chiasmate associations of chromosomes at this stage revealed some specificity in the associations of homologous and nonhomologous chromosomes. Inferences about the nature of synapsis in this mutant were drawn from such observations.

Chromosome Pairing↗

Hybrids and amphiploids of Aegilops ovata L. with Secale cereale L.: production, morphology and fertility.

Hybrids (2n = 3x = 21) between Aegilops ovata and Secale cereale were produced via embryo rescue. Most hybrid morphological traits were intremediate between parents, the plants grew vigorously but were completely sterile. The average frequency of chromosome associations at metaphase I was 19.57-20.19 univalents and 0.40-0.86 rod bivalents. The fertility of the hybrids was restored by doubling their chromosome numbers by colchicine treatment and in vitro propagation. Selfed seeds were obtained from colchicine-doubled sectors and some callus regenerates. The seeds were mostly well formed and developed vigorous plants without embryo culture. Colchicine- and callus-derived amphiploids (2n = 6x = 42) resembled the F(1) plants in overall morphology, but showed a lower tillering ability, broader leaves, thicker culms and larger spikes.

Fertility↗

[Genetic analysis of inheritance of mei8, sy1 and sy10 mutations, disrupting the correct meiosis in the rye Secale cereale L].

It is shown that mutations mei8 (irregular condensation and fragmentation of meiotic chromosomes), sy1 (asynapsis), and sy10 (heterologous synapsis) of rye Secale cereal are nonallelic. In double mutants mei8 sy1 and mei8 sy10 both mutations are expressed simultaneously and independently of each other. A study of joint inheritance of mutations sy1 and sy10 revealed their interaction by means of recessive epistasis: the double mutants has the sy10 phenotype. This means that the sy10 gene controls an earlier stage of synapsis in meiotic prophase than the sy1 gene. Mutation mei8 is inherited independently of sy1 but it is linked to sy10 (recombination frequency 26.8 +/- 3.58%).

Chromosome Pairing↗

[Genetic analysis of the short-stem populations of rye (Secale cereale L.) for the plant height trait].

The process and results of genetic analysis of the short-stem rye populations (Secale cereale L.) for the trait "plant height" are described. The analysis has been performed with the joint scaling test using data of estimation of the parental and hybrid populations developed in the cyclic reciprocal crosses between four initial populations. Monogenic, digenic, and trigenic differences between parental rye populations for two genes of incomplete dominance and one multiallelic gene with additive allelic interaction were determined.

Genes, Dominant↗

[Genetic collection of meiotic mutants of rye Secale cereale L].

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F2 generations after self-fertilization of the F1 hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome "bouquet" was impaired, and all chromosomes were univalent in meiotic metaphase I in 96% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase II. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.

Chromatin Assembly and Disassembly↗

Identification of LMW glutenin-like genes from Secale sylvestre host.

Three low-molecular-weight (LMW) glutenin-like genes (designated as Ssy1, Ssy2 and Ssy3) from Secale sylvestre Host were isolated and characterized. The three genes consist of a predicted highly conservative signal peptide with 20 amino acids, a short N-terminal region with 13 amino acids, a highly variable repetitive domain and a less variable C-terminal domain. The deduced amino acid sequences of the three genes were the LMW-m type due to a methionine residue at the N-terminus. The phylogenic analysis indicated that the prolamin genes could be perfectly clustered into five groups, including HMW-GS, LMW-GS, alpha/beta-, gamma- and omega-prolamin. The LMW glutenin-like genes of S. sylvestre were more orthologous with the LMW-GS genes of wheat and B hordein genes of barley, which also had been confirmed by the homology analysis with the LMW-GS of wheat at Glu-A3, Glu-B3 and Glu-D3 loci. These results indicated that a chromosome locus (designated as Glu-R3) might be located on the R genome of S. sylvestre with the functions similar to the Glu-3 locus in wheat and its related species.

Amino Acid Sequence↗

Localisation of foldback DNA sequences in nuclei chromosomes of Scilla, Secale, and of mouse.

Foldback DNA, prepared from mouse and Scilla sibirica main band DNA, and from rye (Secale cereale) total DNA, was characterised by denaturation, renaturation, and electron microscopy. 3H-cRNA of this DNA was hybridised in situ to nuclei and chromosomes of the respective species. There is no universal labelling pattern among the three species. In mouse, highly repetitive foldback DNA is present in the whole chromatin including the satellite DNA-containing regions. In Scilla sibirica, on the contrary, the highly repetitive foldback sequences are excluded form the satellite DNA loci and are arranged in clusters in the remaining chromatin. In rye, there is a clear preferential labelling of the chromocenters in the interphase nuclei as well as metaphase chromosomes, indicating that highly repetitive foldback DNA is preferentially located among other highly repetitive sequences.

Animals↗